CHROMOSOMES: GENERAL ACcouNT Chromosomes are filamentous. bodies Which us and which become visible during cell dys oal, Present of the genes or units of heredity. Chiesa ‘They are «active nucleus due to their high water conor we "et “uring cell division. Most of the chromosomert: gut sear tsones. In addition there are One oF tvo of sex ms in ae are feterosomes which carry the genes for determination of sex. In the pres a chapter only the chromosomes of eukaryote organisms are being ie with, Prokaryote chromosomes willbe described in the chapter on ‘Bacteria’. jn the nucle the carriers visible in th clearly seen NUMBER OF CHROMOSOMES I the individuals of a species have the same number related species usually have similar chromosome le sets of chromosomes is called euploidy. Itin- Ghies haploids, diploids, triploids, tetraploids ete. Gametes normally tontain only one set of chromosomes. This number is called the ‘haploid umber (n). Somatic cells usually contain two sets of chromosomes (diploid number : 2n). Triploids have three sets of chromosomes Gn), and tetraploids four sets (4n). The condition in which the chromosome sels are present in multiples of 1 is called polyploidy. When a change in the chromosome number does not involve entire sets of chromosomes, baal few ofthe ehromosomes, the situation is called aneuploidy. ee ‘types of ancuploids are monosomics (2n—I), trisomies (rt) mulsomics (2n—2), tetrasomics (on-+2),, double monosomics 1) and double trisomies (2n +1-+1)- wire mat haploid chromosome number recorded in eukaryotes & rears ene tiation) and Ophryotrocka pueriis (polychaete) compound che locephala only one chromosome is found, but this is 2 Atomang chrometome which divides into as many 28 190 chromosomes I. In higher plants only a few species have more than 15 Normally all ofchromosomes. Closely numbers. Presence of who Scanned with CamScanner92 Cell Bion up to 50.are not uncom, numbers a uP jst animals and play” chromosomes (haploid). In animals, mon. The number of haploid chromosomes in most lies between 6 and 25. In animals the highest cae onnneneton number is 127 in the hermit crab Eupagurus send * put thi a reticulatum upto 630 chromosomes have been . int ig probably a polyploid. CHROMOSOME SIZE ies from 1 micron (some fungi) to 3p ‘The length of chromosomes varies FOM | TT cpromosomes of This excludes the 2 mm long. These chromosomes, howeve, astate of metaphasic contra. y be of similar size (sym. microns (Trillium). Diptera, which may be : represent a special case as they f° not in tien, All the chromosomes of species ma! ; metrical karyotype), OF the different chromosomes may Vary in size ie there may either be two (asymmetrical karyotype). 10 the latter case ; distinct size groups (e.g. Yucca varkansana), or there may be @ gradual Series of different sizes (e. 8+ ™man)- Chromosome size may vary greatly in ! : example the chromosomes of Trillium are hundred times the size of the chromosomes of Medeola, a closely related genus. Size differences may of a genus: for example the chromo- be found in the different species half the size of the chromosomes of somes of Allium porrum are one oh e may also vary within the species Allium sativum. Chromosome size e.g. in different varieties. The fly Chironomous thumii thumii bas 21%, more DNA than C.t. piger. Size variation may also take place in different regions or in different stages of the same organism. In the plant Mediola for example, the root tip chromosomes are 50% longer than the shoot tip chromosomes. Also, in certain marine insects the chromosomes of the early blastula are smaller than those of later stage tissues. closely related genera. For STRUCTURE OF THE CHROMOSOME In earlier light microscopy descriptions the chromosome, OF chro" matid, was thought to consist of a coiled thread called the chromonen lying in a matrix. The chromosome was supposed to be covered by? membranous pellicle. Electron micrnscope studies later showed there is no definite membranous pellicle surrounding the chromos? Other structures present in the chromosome include the centrome™ art fomsritlon, nucleolar organisers, telomeres and satellité . 6.1 al eo | and .2). The structure of the chromosome will now be ‘ Scanned with CamScannerGeneral Account 93 ot sores B-4—chromonemats Telomere {—Chromomers Antercatary heterochromatin Secondary ‘constriction It constiction Primary }-—constriction (centromere) Nucleolus Seconda v Sseamieton | constriction | B) treat congenic etl Constitutive heterochromati ‘Satelite oe Fig. 6.1. Structure of a typical chromosome, Fig. 6.2. Model of constitutive heterochromatin in the metaphase chromosome of a mammal, Chromonema. Under the light microscope the metaphasic chromo- seme eppears to be made up of two subunits called chromatids. These : tees twisted around each other, If the chromosomes are treated ag oe Proteins, each chromatid is seen to consist of "naa, ‘There i Possibly the subchromatids Tepresent the chromo- romonemata a ait no general agreement regarding the number dS have ben iggeaicd romosome. From 2 to 32, and even more, toner Tee Various chromosome models ome kinetochore cae has a constricted Tegion called the Ian is constant ‘c cine constriction. The position of the of th eae; Particular chromosome. The structure and s different from that of the rest of the chro- Stra centro, 4 Scanned with CamScanner94 Cell Biology pace During division the centromere is functional, while the ya, TOmMosome is genetically inactive. Therefore, the Centrome, may the locus of genes for mitotic and meiotic activity. The position the centromere varies in different chromosomes. Since the spindle fibres are attached to the centromere,the shape of the chromosome durin, anaphasic movements will depend upon the position of the centromere Four categories of chromosomes are recognized, depending on thy position of the centromere. These are called metacentric, submetacentrie, acrocentric and teloceniric chromosomes (Fis. 6 3) IIL | | Aciocenttic ——-Telocentric Tie. 6.3. Types of chromosomes based on the position of the centromere. (1) Metacentric. If the centromere 1s near the middle of the chro- mosome (median),the two arms of the chromosome are nearly equal The chromosome appears V-shaped during anaphasic movement, Such a chromosome is called metacentric. (2) Submetacentric. Ifthe centromere is situated some distance away from the middle (submedian), one arm of the chromosome will be Shorter than the other. Such a chromosome will appear L-shaped during anaphasic movement and is called submetacentric. (3) Acrocentric, When the centromere is situated near the end of the chromosome (sub/erminal) it appears rod-shaped and is called acrocentric. (4) Telocentric. If the centromere is truly terminal, i. the tip of the chromosome, the chromosome is said to be fe Telocentric chromosome are very rare. Chromosome IV of D) was thought to be telocentric until the discovery of a very short secon arm showed it to be acrocentric. Truly telocentric chromosomes have been reported by Marks (1957) in some species of the plants, Protozoa a" mammals, and have been confirmed later by the electron microscope In Trillium and Tradescantia all the chromosomes are metacentt In some grasshoppers all are acrocentric. In man three types of che oe found: metacentrio, submetacentric and acrocentri¢ (73 e. situated at Jocentric. rosophila Scanned with CamScannerCell Bi, 96 lin e ha Polycentric chromosomes. Generally the Te aa ie only ong centromere. However, in some animals chemi eran and homopie insects, Ascaris) and plants the centromere sath of the chromosome sina di ion along the len, aH but lies in a diffused condition i eae acalin such cases the centromere is called a diffuse centrony aie me is said to be polycentric. / ; Di tric chromosomes with two centromeres are sometimes produce, fea f structurally different homolog, le during pairing of . BOus EG eee (produced as a result of translocation). If these two Centro, I ir ¢ chromosome meres move to opposite poles during eae oe ae resulting Rarely, a new centromere may appear ntroméé: Neo an abnormality. Such a centromere is called a neoce! 7 centro, meres have been reported in Zea mays cultures. ‘Acentric chromosomes. Sometimes a chromosome may undergo break into two, so that only one part has the centromere while the othe, is without the centromere. The part lacking the centromere jx called the acentric fragment. It doesnot take part in mitosis, as spindle fibres cannot be attached to it. Its loss usually results in lethality. Arm ratio. The ratio of length of the long arm to the short am of a chromosomes called the arm ratio. It is characteristic for each single chromosome. In chromosomes where the centromere js neatly terminal (e.g. acrocentric chromosomes) the arm ratio is high. Chro- mosomes in which the centromere is located in or near the middle (metacentric and submetacentric chromosomes) have a low arm ratio, The arm ratio is useful in distinguishing chromosomes which are similat in total length. In humans metacentric chromosomes have arm ratios ranging from 1.07 to 1,95, submetacentrics from 2.05 to 3.75 and acto centries from 5.00 to 11.94(Vogel, 1961). __, Centromeric chromomeres. In metaphasic chromosomes, which coo ist of two chromatids, four granules can be seen within the centromett These granules are called centromeric chromomeres, and are about 05 ee in size. They are arranged in a square. During anaphase, whe? Pawiraee eee a (chromosome) oe “ centromere occurs. The centromere iy eens duplication oF Tegion of the chromonema, Itis @p See ee ae Possibly the formation, egy, SBeealized for the attachment, fibres can be seen extendin fror le fibres. During cell division spi! Secondary constrict 8 from either side of the centromere. ictions, to. Scanned with CamScannermes ; General Account Chromosol 7 pough some cytologists also refer to the nucleolar ee secondary constriction. The location of the the js constant for a particular chromosome, is M entifcation of chromosomes. It has been suggested that the secondary ‘ nstrictions represent sites of breakage and subsequent fusion. cor . In man pmmgary constrictions II are found on the long arms of chromosoncy organiser ag © secondary constriction and is therefore useful for 11, 13, 16 and Y. Nucleolar organiser (secondary constriction 1), Normally in each roid set_of chromosomes, two homoglogous chromosomes have dine nal constrictions’ called nucleolar organisers. ‘These are so called a they are necessary for the formation of the nucleous. The beens is formed in the post-mitotic reconstruction phase. Under the nuctmperoscope the nucleolar organiser appears as a ‘constriction” near bs end of chromosome. The part of the chromosome beyond the qucleolar organiser is very short and appears like a sphere (satellite). In jumans,chromosomes 13,14,15,21,22 and Y have nucleolar organisers and satellites. Chromosomes bearing satellites are called _SAT-chromo- tomes, The prefix SAT stands for ‘Sine Acid Thymonucleinico’ (without thymonucliec acid or DNA), since the chromosome on staining shows relative deficiency of DNA in the nucleolar organiser region. There are at least two SAT-chromsomes in each diploid nucleus. Nucleolar organisers have been reported in several animals and plants, and are probably of universal occurence. Each nucleolar organi- ser may be associated with a nucleolus, or both nucleolar organisers may beattached to one nucleolus. In some diploids and in polyploids the number of nucleoi and nucleolar organisers may be more than two. Many polyploid species, however, have only two SAT-chromosomes, for example hexaploid wheat, The nucleolar organiser Tepresents about 0.3%of the total amount of ieee DNA. It, therefore, has several gene loci, which are believed ds cerned with the formation of 28S ribosomal RNA. ‘omeies. The tips of the chromosomes arc called telomeres. The TABLE 6,2 Other Term Metacentric Metacentric Submetacentric Subtelocentric Acrocentric* Telocentric Scanned with CamScannerCelt Bi, 98 ell Pol, telomere differs in structure and composition from the Gee of be chro, Some. Ithas a unique property in that it prevents he ends of chromosomes from sticking together. If the chromosome ends mh. the ate broken by X-ray irradiation the newly created ee hey Uniderg, fusion. A telomere will not, however, unite with a bro) - Nor vil two telomeres unite. Telomeres ‘are épecially modified regions of the chromosome for attachment to the nuclear envelope. The ends of y,° chromosome are associated with the nuclear envelope from telophase to prophase (i.e. through interphase). a Earyetce A a of chromosomes, ofan individual or species called a karyotype. Inman the 23 pairs of chromosomes IN Somatic ce, form the karyotype. It is possible to identify individual chromosomes o, the basis of the following characteristics : (1) the total length of the chr, mosomes, (2) arm ratio (ratio of the lengths of the long and the Shor, arms as determined by the position of the centromere), (3) the position of the secondary constrictions and nucleolar organisers and (4) subdivision of the chromosome into euchromatic and heterochromatic regions. Hom, Ologous pairs of identified chromosomes can be arranged in a series op decreasing lengths. Such an arrangment is called an idiogram (Fig, 64), In some species the chromosomes of set may be of the same length and have their centromeres located inthe same position. In such cases it is not possible to prepare idiograms. ’& GROUP 8 GROUP 289 ou ix These chromosomes « ipernumerary chromosom” Scanned with CamScanner: 1 Account nosomes : General ~ chrom \ accessory chromosomes or B chromosomes, Supernumerary chromu- ames arenot homologous with any of the normal chromosomes, ‘and so . , they do not form synapses with the latter, They, however, form synapses peng themselves. Supernumerary chro am : osomes are not present in all individuals of a species but are confined 10 a few individuals. They the generally smaller than normal chromosomes, They are found are commonly in anit more in plants than in animals, supernumerary chromosomes do not usually have any effect onthe oupeand hence are genetically unnecessary. In some plane eens Pere pernumerary chromosomes esult in decreased vigour. All ew eof extra chromosomes in a few individus of a species are aa feapetauimeratyfohromasanes The extra chromosome Le s (for example trisomy of chromosome 21 in man, aoa syndrome) is not considered to be a supernume: ence itafects the Phenotype. Among animals 5 Spowosomtes have been reported mostly in insects, Of the 50 species of | Sw homed grasshoppers belonging to the family Acrididae. Super. numerary chromosomes have also been reported in two species of Hat voms. Inplants these chromosomes have been reported in sevens] species, mostly Angiosperms. Usually each nucleus has one or two supernumerary chromosomes, In Tradescantia edwardsiana there are 5 supernumerary chromosomes in addition to the twelve somatic chromosomes (Fig, 6.5). As a result not in which results tary chromo- upernumerary C sy > Seeeacaoe 1 ‘upernumerary chromosomes (5) eraries per nucleus ; ae in ti iOpen may be eliminated from certain tissues or a tu ™may persist in others, For example in Dee th ete aminated from the root by ed to the next generat have been obtained ition, Scanned with CamScannerCell Biol, 100 elieved to be derived from ay, moet romere and the hetern, . cent! ied arms on either side of y, Supernumerary chromosome: somes (Fig. 6.6). They have rel chromatic genetically inert parts of t wo fe Heterochromatin Hateroehromatin c ~<¢ Euchromatin fF Euchromatin osomes. The euchromatic part of rary chrom pupernumeraty cheoPromatic ,part is retained as the Fig. 6.6.Derivation of 8 ‘The heteroc! the autosomes is lost. supernumerary chromosome, C-centromere, a. centromere. The distal euchromatic parts of the arms containing genes have, however, been lost. LL CHROMOSOMES chromosomes are so called because they They are found in the family The germ line cells in females Limited chromosomes or L are large and limited to the germ line. Sciaridae (Diptera : Insecta) (Fig. 6.7). COG) OG) Somatic cal call Sat “ar ct 8 10 chromosomes 7 9 Fig- 6.7. Schematic representation of L chromosomes in the Sciaridae. have i ret desert » (three pairs of autosomes, a pair of X chrome fone: and pir of Lehromosomes). ‘Those of males have 9 chrom chromosomes in females and Tis male. he Doonan ‘ce cromsomes in female and7 in males, the L chromosomes bei eliminated from nuclei deine tenn, “eat aBes,_E_ chromosomes a inthe germ line cell lestined to form somatic tissues, but are retained chromosomes in than L chromosomes differ from supernunl they are constant in all individuals of the sect 501 having them, sa 1. Supernu: individuals of the Spiga chromosomes are found only i Scanned with CamScannerchwmosores General Account tor m CHROMOSOMES chromosomes ot minute chromosomes a xtremely small size (0.5 micron or less). They have been reported species of bryophytes and in insects of the family Coreidae reroptera)- They have been seen mainly during meiosis, and only (Hel jonally during mitosis. Usually one or two chromosomes are seen, vryo Say also be present. That these bodies are chromosomer the fact that they contain DNA and undergo Pairing. The er, separate by metaphase Tinto two univalents, A quadri- = group may also be formed, with the four units connected together parti cee d inarectangle. Inthe moss Sphagnum there are 19 large — a and two m chromosomes (Fig. 6.8). Because m chromosomes i re 80 called because of | nein jn many 19 large bivalents ‘Second m chromotome (quadripartitey m chromosome ‘consisting of univalents Fig- 6-8- Diagram of m chromosomes, arepresent in all the individuals of a species in which they are found they are not considered to be supernumerary chromosomes. S aud E CHROMOSOMES Sand E chromosomes have been reported in insects in the family Cecidomyiidae (gall insects) and family Chironomidae (Diptera). Ii ‘he gail inseet Maistor both males and females have 48 chromosomes in ferm line cells (Fig. 6.9). In somatic cells, however, there are only 12 © Eliminates 7 a= pont” F ecnesona B- 6.9, Sche Uallinsect Adiasne tion of the S and E chromosomes of the | Malic repre, Scanned with CamScannerCell Biot, 102 chromosomes in females and 6 in males. anes eats hang been eliminated from somatic cells of females a The wage stages of the embryo, AU, iminati during clea elimination takes place 8 im tine cells. Chromosomes wi chromosomes are retained in the germ ee s are cal emote are present in both germ and somatic cell i pr which are eliminated from somatic cells but are present in ger cae called E chromosomes. ‘Thus in a el ae eel haye 12.8 chromosomes and 36 E chromosomes. In mie S000 ne oxy there are 6 $ chromosomes and 42 E ee ae m7 ez EON etc half its $ chromosomes from each parent, W! ile Somey are received from the female parent. MEGACHROMOSOMES Certain large chromosomes, upto 15 times the length of norma chromosomes, have been described inafew species of hybrids, f example Nicotiana hybrids. These chromosomes have been calle, megachromoscmes. They are not to be confused with the giant chromo. somes found in the salivary glands of dipterous insects. Giant chromo. somes are polytenic, i.e. they consist of many strands, Megachromosomes are found only in a very small proportion of the total number of cells. Usually there is only one megachromosome per cell, although as many as 7have been found. They are largely heterochromatic. They may have a single centromere or may be dicentic or acentric. Megachromosomes appear in successive generations although they are not transmitted through the gametes. This indicates that the ability to produce these chromosomes is inheritable. HETEROCHROMATIN AND EUCHROMATIN Certain segments of the chromosomes, or the entire chromosomes, are more condensed than the rest of the karyotype during various stage of the cell cycle, Such difference in thickening has been called heter” Pycnosis. Heteropyenosis may be positive, where there is overconde” sation, or negative, where there is undercondensation. Paces which remain condensed during interphase are cll S, €.g. the sex chromosomes of insects. The ™ condensed chro; . ied mosomes whic ing i e_ call cuchromosomen h extend during interphase ar ati Chroma trials of two types, heterochromatin and eel heterochtomati at*Tial showing heteropyenosis at any stage is tin. Regic Fe hete a Regions of the c} sch nevel “roPYcnosis consist of euchtomatin hromosomes which Scanned with CamScannerymes : General Account chromeso : 103 ‘There are several differences between heterochromatin and euchro. in. + . ot) Heterochromatin stains deeply while euchromatin stains less seer Heterochromatin is found in the condensed regions of the chro- ome, andis associated with tight folding and coiling of the eh mmostigbre. Euchromatin consists of the diffused or less tightie ney som It undergoes the typical eycle of condensation during vitae ey gecondensation during interphase, _ @) Heterochromatin is late teplicating. It replicates at the end of e § phase of the mitotic cycle. Euchromatin replicates during the early rage of the S phase. “ (4) Heterochromatin does not become acetylated. Euchromatin take up acetic acid (via_ acetyl CoA) on its histone during interphase, (6) Heterochromatin is more labile than euchromatin and is affected by temperature, SCX, age of parents, proximity to the centromere and presence of an additional Y chromosome. © Heterochromatin is Telatively inert metabolically. Hetero- chromatin segment contain relatively few genes in relation to their length put nevertheless a few genes are present. In Drosophila melanogaster the Y chromosome is entirely heterochromatic, Males lacking the Y chromosome have a normal external phenotype and are viable. They are, howeve, sterile, because normal sperm formation does not take place during spermateliosis. The supernumerary chromosomes of maize are also examples of inert heterochromatin. They may be present in varying numbers without affecting the phenotype. The DNA of heterochromatin is genetically inert and does not transcribe messenger RNA for protein synthesis. Euchromatin on. the other hand is genetically active. Its DNA synthesizes messenger RNA-during interphase. (1) The crossover frequency is less in heterochromatin than in cuchromatin. This is apparently because condensed regions of the chroy a fibre cannot come close together for frequent crossover. This esses may help in protecting vital genes from the effects of ‘ aan tain types of heterochromatin, constitutive hetero- ows heteropyeenat heterochromatin, Constitutive heterochromain other hang nos i all cell types. Facultative heterochromatin on is hetero, i i i Some parti pycnotic only in some special cell types, or at Patticular stages of the life cycle. ° ° ‘onstiturj (DNA), This neterochromatin was originally called satellite DNA NA is highly eit insetive during protein synthesis. Constitutive Petitive. It consists of comparatively short identical Scanned with CamScanner104 oa Biology 4 several hundred thousand times, genes (DNA) which are repeate feribe messenger RNA for se enes can replicate but do not tran: 7 Protgi Synthesis. They are therefore inert and are partly eee Consing tive heterochromatin is generally found in blocks, usually around ar, such as the centromeres and secondary constrictions. is Possible Function is to give centromeric strength and to act as spacer tween vial peg It provides points of attachment with the nuclear meml br ne an Sitest Tecognition and association of homologous chromosom: juring Meiosis Constitutive heterochromatin is seen consistently in the same Tegions of both homologous chromosomes of a pair. Facultative heterochromatin comprises about 2.5% of the genome, It is metabolically inactive. Facultative heterochromatin becomes the sey chromatin body early in embryogenesis. It is the heterochromatin which results from inactivation of one the two X chromosomes in females, Some workers have suggested that heterochromatin and euchro. matin represent different states of the same substance. According to them a chromosome segment may be heterochromatic under certain conditions and euchromatic under other. This belief has however, been chailenged by Yunis and Yasmineh (1972). These workers have found that the giant chromosomes of the European field vole Microtus agrestis show condensation in interphasic nuclei throughout development. Types of chromatin in different regions of the chromosome. (1) Telomeres. In some organisms evidence has been presented that the telomeres or ends of chromosomes contain heterochromatin ot Tepetitives DNA. The evidence has been given mostly for plants. The telomeres Probably serve as chromosome ends, maintaining the individual integrity of each chromosome, @) Primary constriction (centromere). The primary constriction o the centromere contains chromatin to which spindle fibres are attached: {tis continuous with heterochromatin on both sides, It has been sto¥? (se Rac 1970) that the heterochromatin is of two types, called « and ae vomatin by Heitz (1934), The heterochromatin around tht romeng (2) a Compact and resistant to destaining, ‘The rest of centromeric heterochi in is i i ; suooete romatin (A) is relatively diffused and less resist e 3) . oler o Secondary scisttiction I (the nncleolar organiser). ‘The 1» Contains mee cuchromatin and is imagined to lie withio ns the genes for the synthesis of 188 and 28S ribose mal RNA. RNA synthesi during mid-mitosig, * #®S Place throughout the eell cycle, 2° Scanned with CamScannercromosomes General Account ia oe secondary constriction II. The scondary constriction II consists chromatin which carries out MRNA synthesis almost throughout the euciysie, like the nucleolar organiser. Tt has been suggested that cat some cases the secondary constrictions If may represent sites in at’ josomal RNA synthesis. of SO satellite. A mass of heterochromatin at one end of the chro- °) represents the so-called satellite, Fuchromatin. The bulk of the chromosome is made up of matin. It is believed to be partly composed of non-repeated DNA exshomat synthesize mRNA during interphase. sequence Intercalary heterochromatin (represented by dots and short igure) is found within the euchromatin. segments i the fi CHROMOSOME MODELS ished that the chromosome contains DNA and ‘The question however remains: how is the DNA arranged in the chromosome ? Several models have been proposed to explain the associa tion of DNA and protein in the chromosome, There are three main areas of controversy in the various chromosome models. (1) Does the chromosome contain a single strand or multiple strands of DNA ? (2) Is the DNA present as a continuous strand from one end of the chromo- some to the other, or is itinterrupted by ‘linkers’? (3) How is the protein associated with the DNA molecule? The requirements for an acceptable model of the chromosome are:(1) that the linear sequence of genes should be preserved, and (2) that replication and segregation of DNA should be explained. The various chromosome models may be grouped under two heads: mulipl strand models and single strand models, MULTIPLE STRAND MODELS The i es fe pale strand models are based on the fact that cytologists Frits ae ae strands of varying thickness by different techniques. "ios electron i 30 to 200A, and even SOOA, have been seen by mall stand mel fees during interphase and mitosis. In the D “Potcn strands ‘ chromosome is supposed to consist of several Pi t that each dine on on radiation damage to chromosomes ma, ie. two cosa of at least four subunits (chro- Stange Wan Models peabent each with two halves (Nebel, 1938: : CEN Sugvested, Tock the existence of 4,8, 16 and 32 la each double heli Strand or fibril is supposed to contain Chromatig +. nclX associated wit ; i mat is reported t te with Protein. Thus in Trades- ‘© have eight 200A. fibrils (16 in the Scanned with CamScanner It is well establ protein.| te Cell Bing chromosome). Also, according to Berger (1938) puelcrel (1946 in mosquito prophase chromosomes contain at least 16 if not 32 stron? Friedrich-Freska and Handewitz (1953) considered the chromoso, of Amoeba proteus to be 16-stranded, on the basis of a study 9 active decay of chromosomes. Simple multi-stranded model (Steffensen, 1952, 1961). Accordin, this mode! the chromosome consists of 64 double helices of DNAarrae® in a parallel manner, and twisted together like the strands of a rope, 7 difficulty with this model is that replication of DNA would involve large amount of untwisting and untangling. Ris’ multistrand model (Fig. 6.10). According to this model 4, 20A wide DNA molecule is associated with histone (Protein) to fom, Mes of f radig DNA Protein Elementary chromosome fibril Nucleoprotein 100A 200A Fibril Chromomere segment Chromosome Fig- 6.10. Ris’ multistrand model. 40A DNA-histone (nucleoprotein) fibril. Two 40A nucleoprotein fibts make up a 100A fibril called the elementary choromosoime fibril. (R 1360, 1961). Two 100A fibrils wrap around each other to form a fibril. Thus each 200A fibrils contains four parallel DNA-histone db helices. The 200A fibrils are associated to form the chromatid © chromosome. Each ch: romatid aj is element chromosome fibrils. Ce aie pee evidence for this model was based on observations of elt? ieee of chromosome extracted with citrate and other chee ‘as, however, shown that the inter-twining of fibres ot wi Was a secondary Phenomenon due to extracting procedures. Late! (1966) led him to give up this model. M . wit Pa ie Gvidence now indicates that chromosomes are 3° ‘atement applies tonormal chromosomes. A tYP Scanned with CamScannerrosomes : General Account rom os 107 Mi somes, called polytene cdromosomes, may have several hundred chore These stands are, however, formed by division of chromatids, s. stra Single Strand Models jous studies have demonstrated that chromosomes are single Varionnese studies include the stretching of lampbrush chromosomes stranded. These of enzymes on their structure, Experimental evidence and ae ged by damaging chromosomes with X-rays, and by labelling is also Pore eecomets The general conclusion is that the chromo. duplicating one coiled or folded DNA molecule. some s Og side chain model or centipede model (Fig. 6.11). Taylor Taylor ‘eed that the chromosomes consist of a long protein back. (199) Posh DNA coils branch off just like the legs of a, centipede. bone from iy PON ON ale DNA. ———{, 6-11 6-12 HE: 6:11. Taylor's centipede model Fig. 6.12. The Freese-Taylor model. Scanned with CamScanner108 Cell Bote He explained replication by assuming that the protein backbone js layered, and that these layers can be pulled apart. Each layer woy have one strand of the DNA helix on separation. Anew chromatid con then be built up on each separated chromatid. This a lel does oy reconcile with the fact that the genes arc arranged in & fae Sequeno, It is also inconsistent with genetic Cae Sata Stricy, speaking many DNA strands are present, but since # a nm nl from linear backbone this model has been included in the aaa tran Models, ‘The Freese-Taylor model (Fig. 4.12). A secon ‘C9 58) * Propet by Taylor (1959) based on one suggested by Frees aa ee to this model there are two protein spines instea or on “in he Di chains stretch between them like the steps of a la ae effect the the DNA molecules are kept in position by the protein, linkers i the linkers become closely apposed they would form the axis 0! side rome. some, and the DNA would be in the form of lateral loops. h is. mode has the advantage that it can satisfy the concept that the genes ay in linear a order. wend coll model ‘According to Nebel and others the chromoson, is made up of only a single fibril. This fibril is tightly coiled and th il is thrown into secondary coils, a Tis modied model (Fig. 6.13). Ris (1967) suggested a modifi model according to which the chromosome fibril is folded to form te + peotein tot = —, 208 1008 4 on DNA ‘wucleoprotein Fibail , Fig. 6.13 Ris’ modified model. i) Gtromosome. The 20A wide helix is associated with histone (Pr taker ng QUA diameter nucleokistone fibril. Folding of the 100A fbr Wig nets Of calcium bridges to form a 250A diamelt yg 's postulated that the 250A basic fibril undergoes stil Scanned with CamScanner109 Electron microscope pictures of 100A reading chromosomal material at an ipsomnes ! General Account hrom i form the chromosome. fold obtained by Ris by spi interface. ; ied bre model of DuPraw (Fig. 6.14), mounts of human leucocytes under t} sisils sinovate Folde! DuPraw (1965, 1966) studied whole he electron microscope, Metaphase Fig- 6-14 Folded fibre model of DuPraw- He found that sister chromatids consist of irregularly folded fibres 200 to SA in diameter. Since very few or no free fibre ends were found,it ¥s concluded that each chromatid consist of a single fibre (Type B. fibre) folded both longitudinally and transversely), Autoradiography experi- ‘ents have also indicated the existence of a single fibre. The details of chromosome structure according to the DuPraw model The 20A DNA double helix;.56 microns long, is spirally fir tote on m to form a fibril. This fibril if coiled would forma in diameter and 7 to 8 microus long, This fibre is called rl of a The DNA is Packed inside the Type A fibre ina pack- allpacked as: | (The packing ratio is the ratio of the extended The Type A fibre is then in turn coiled in a 1 to foy i in di (packing rao? £1 8 Type B fibre 209 to 250A in diameter, 79280 Tyee 39 °f DNA in the Type B fibre averages 56:1, The i Nhs been fibre ig extensively folded : *, Lape gem’ to form the chromatid, Th y Dermott (1968), Abuelo and Moore 0) Beeson edric (1969) “eDna RS Features DuPraw's fe ere} W's fold "is packed in z led fibre model are that : into a 80-19 OA Type A fibre, and (2) Scanned with CamScannerfo i10 Cell Biot, shat the fibre is further coiled to form a 200-350A Type B fibre. Lampe, (1971) supports the folded fibre model. Kihlman (1971) also suppor the model, stating “The findings. indicate that a mitotic chrom some is formed by coiling and folding of a fibre consisting of a long single DNA molecule associated with protein”. DuPraw’s folded fibre model has ey ae I aero h appeared in several text books. In view of this i include the: comments of M.J.D, White, In his book ‘The Chromosomes (1973) he writes : “Two general hypotheses of chromosome structy, which are certainly incorrect are the ‘folded fibre’ model of DuPraw ang the same author's extraordinary idea that the DNA of all the chromosom, is really continuous, i.e, present in the form of a ring. The “folded fibye model is certainly incompatible with the constant sequence of chrom, Omeres, constrictions, heterochromatic segments etc, along the lengi, of the chromosome, as well as with all observations on the giant lamp. brush and polytene chromosomes and the whole theor; chromosomal rearrangements”. DuPraw's model of DNA-histone association unlikely by the discovery that DNA itself is looped aro to form nucleosomes. Y of structaral is now considered und histone beads Nucleosomes. The chromosomes of eukaryotes are made up of Unphosphorylated H1 Histone core i H2aH2bH3Ha Linker ONA — Exonucleolytic 1200 bp digestion 450 bp Nucleseomes Trimeric , Cor article. Nucleosomes © corse with linker DNA +, The nucleosome. A ‘¢ ‘ith phosphorylated Hy (7 B Ghromatin with Unphosphorylatent De Arporsinleesomes with lindker spec” ‘d Inodel ofahe Mucleosome (Kornberg, 1974). The sent With the linker regiti"0N€8, Han, Hb, My and Hy. Hh Is 8950 Scanned with CamScannersomes : General Account chrom nm fein material called chromatin, The nucleoprotein consist 1 eid, which is DNA, and proteins w\ af muclele types of sperms protamines are In rly (1978) showed that under the elecer svoodcork as a ‘string of beads’ structure and ig my appeats Og umits. These units (the *beads’) hi; of repeat 15). ‘The nucleosome concept re snes (FB: del of DNA-protein association, - mesome a some is an oblate particle about 50-5 seem mince of his : in dame Smeter, around which is wrapped BWA. Ta total weight of sta Oe eel that of DNA. When chromatin is in a partly ue Hstore the diameter of the beads is reduced to about 70A. Such ed state, 7 ects areal mu Bodies, jated with chromatin tiles cf histones have been found associate with chromatin, Fi Ha and’ Ha.” OF these the location of H is believed to Bi, Has, HO, H that of other histones. The histones appear to be eer ni two regiong! One ‘containing & molceules of H2a, H2b, we eer eeeanenieel (Fig, 6.16). The Histone sore ao cols of the octamer of 8 histone molecules, arranged in the believed to consis| f H2a, H2b, H3 and H4, stacked one on top CEE Hecate gee ach of the four types of foam o leosome contains two each o| yp a ee tin experiments, if only three of the four histones an ”. tes, sae Poe are formed. Addition of the fourth are mixed Loy s . osomes. histone at once results in the formation of nucleosom Pe chromatin ade up of a number ‘ve been called nucleo. Presents the latest chro- ‘SA high and 110A, ones, 40A high and Histone core 624 109A A 8 . Fi 6.16, Model of a core Particle. (After Lilley et al 1978), DNA and histone core in side elevation. The same in plan. ; Pictorial view of elevation. listones Luding rats tail H2b, HB and tig ‘ails’, ‘arms? Onsisting of NH,-terminal regions) "sidered to yee? HOWever, been questioned, and histones Fred to be fully globular, Scanned with CamScanner— 112 Cell Biology Yeasts do not have the mammalian equivalent of the histone yj, but possess the four other histones. ‘The macronucleus of Tetrahymeng has all five histone types, but the micronucleus lacks. the equivalents o Hi and H3. In nucleated erythrocytes the amonnt of HI is Teduced, put another histone H5 is present. When the chromation of DNA breaks into fragments of al eaves DNA betw f nuclei is digested with eudonuclease thy out 200 base pairs (bp). It is believe that the endonuclease cl een the nucleosomes. Values of the number of base pairs in the nucleosomes of different organisms show a wide variation from the 200 bp mentioned above, ranging from 154 (Aspergillus) to 241 (see urchin sperm). Further digestion of the nucleosome produces a ‘core particle containing the histone octamer ‘and about 140 bp of DNA. This 140 bp DNA length is common to all types so far investigated, and probably represents the basic level of organisation of chromatin. The nucleosome core particle is a flat structure having the dimensions 57 x 110 x 110A. The DNA linking the core particles is termed linker DNA. Its length is 15-100 bp, depending on the cell type. The linker DNA is coiled or folded in the normal state of chromatin. It can be easily pulled out under tension. Electron microcrographs of such chromatin fibres show the particles some distance apart. Available data suggest that the DNA is located on the outside of the nucleosome, and that it is in the B structure, It is assumed that the DNA double helix is wound around the histone core in a superhelix (Fig. 6.17). It is estimated that there are 75-82 base pairs per tum of the supernelix. Thus for the 142 bp there are about 1% tums of the superhelix around the histone core (Fig. 6.18). The DNA double helices are approximately in phase on the two turns, bringing the phos pate groups periodically close together. The pitch of the DNA tum i small enough to permit interaction between the two turns, This inter action is mediated by cations and/or histone salt bridges. When chromatin is digested bi A i y the nuclease DNase I, the DN poe up in to lengths of 10, 20, 30, 40 bases etc. This suggests th a oN in ee eeeoms is available to cleavage by nuclease atl . ¢ sites most readil 0, 100, 120 and 130 bases from the 5’ ia er eraarel esis o® The hi , some in epproxia and DNA are assumed to be arranged in the nucle aa a bipartite mirror symmetry, This is evident fro” v ucleosomes split in half forming two sseminucleosom during dialysis agai ‘ gainst water, (2) D: . ay intervals by DNase II under a ea is cleaved S. Scanned with CamScanner113 perhelix in the nucleosome core. ® . tic representation of DNA sul 2 ! we" Sebemsrtbe euperlix is 28A and the average diameter i 90 A. (After 4 Klug et al, 1978)» ‘ . n d 4 hr 4 (a) (8) «c) 4} Fip6t8, Mode showing appearance of 12 turns of the DNA superbelix (After af Klug et al, 1978). It will thus be seen that digestion of chromatin takes place in three | steps: (1) cleavage of DNA between the nucleosomes, (2) degradation of nucleosomes, to core particles in which linker DNA is converted to acid soluble form, and (3) cleavage within the core particle. Arrangement of chromatin in chromosomes. Mitotic chromosomes mene, up by the coiling of a large cylindrical fibre, the unit fibre. a et ca to be formed by three levels of coiling (Zeuthen LT re ea ; ee ene et cling of DNA is in the string of nucleosomes. : Set closomes is then coiled into a 300A diameter . The solenoid i . id is further coiled into a super solenoid structure with a ‘ameter of 4,009, Solenoi n000A, 5 4 - Sid structure fe a eral 300A thick (Fig. 6:19). This super . Scanned with CamScanner4 Fig. 6.19. Diagram showing cross section unit fibre. The contraction ratio at each level of coiling would be 7, 6 and 30-40 (7 for the string of nucleosomes, 6 for the solenoid and 30-40 for for the super solenoid structure of the unit fibre). Thus the over | contraction of DNA within t 1,500 fold. The unit fibre microscopy. factor of about 7,000. The solenoidal model for the superstructure in chromatin was fint proposed by Finch and Clug (1976). CHROMOSOME PROTEINS In some virus and bacterial cells the negatively charged phosphalt groups of DNA may be neutralized by positively charged polyamines divalent cations like Mgt+, up of DNA and protein, charse of DNA is neutralized by protamines, In the somatic cells ryotes the neutralizing proteins are histones. In addition to histones several nonhistone also found in chromosomes, Protamines are sim, ple (~ 4,000), They are very are found in th It is further coiled in the chromosome by a factor of about 5, The final contraction of DNA in the chromatid is therefore bya © spermatozoa of some fish, (e.g. salmon, and he of the super-solenoid structure ofthe fhe unit fibre would be in the order of 1,300- corresponds to the ‘chromonema’ of light The chromosomes of eukaryotes are matt In the spermatozoa of some fish the negttit | chromosomal proteins (NHC_proteids) Protamines basic Proteins with a low molecular welll! Tich in the basic amino acid arginine. 1) sci Scanned with CamScannerjomes ; General Account Chromos¢ tis ails and squids. The protamines appear . DNA and are tightly bound to the nuclele atte Ph teed tides of protamines generally consist of 28 amino acid residues of whieh 19 are arginines and 8 ord nonbasic amino acids. Because of their jimited ‘amino acid constitution there are not many different protamine types: possibly there are only three distinctly different molecules. fowl 0 ground Histones Histones are the small basic proteins associated with the DNA of cells. They are the major general structural proteins of nd also act as gene repressors. The DNA-histone ratio in ry of plants and animals is 1:1. The different histones have been distinguished by amino acid sequence criteria. There are five | beetipal classes of histones, H1(H3), H2a, H2b, H3 and Hé,on the | Pircof the Ciba Symposium nomenclature, Histones are rich in the basic amino acids arginine and lysine, but t completely lack tryptophan. They are very highly modified proteins, | the modifications including acetylation, methylation and phosphorylation. Histones are very highly conserved proteins, with very little variation in their amino acid sequences. : On the basic of their relative contents of ] § 1 fall into three groups. | j chromatin @! wide variet | arginine and lysine histones i). Very lysine rich (H1) ii) Lysine rich (H2a, H2b) iii) Arginine rich (H3, 4) Table 6.3: Characteristics of histones. Clary Type and Lys/Are Molecular ‘Total ~—‘N-ter~ , ratio weight Residues minal C-terminal Very lysine rich 21,500 215 AcSer Lys (22.0) Usine rich 14,004 129 AcSer Lys (in) Lysine rich (2.50) 13,774 125 Pro Lys Arginine rich ron 15,324 135 Arginine rich 079) Uze4 Histon Tay © 1 (as) F Y Vary as much A tae 21,500) is the most divergent histone and by amino acid substitutions among the Scanned with CamScannerCell Bio ony | 116 / ; st species-specific ini subfractions of agiven organism. Bi ach Pith different amin’ sequence. One to eight subfractions rigferent § species. The protein , een observe Tis 22.0. THE proten acid sequences, have been ved yery lysine rich. The Iysine/anBinine Tar ate Ceterminal. has Ac-serine at the Sw br ond 13,71 spective are iy : 7 7 0, respe . Ha rich Histon inearginine ratios 1 id bad Ee aieTal is Aeserin He NT es cose an cod Hi and ee jas lysine. H evolved at a more rapid rates ; Histone 3 (MW 15,324) is @ highly © arginine. ‘The lysine/arginine ratio is 0.72. conserved protein, rich in Alanine is present both at als have a major H3 the N+ J and C-terminal Many mam i inal. am terminal ai a a am ie component containing 10 © ssteines at positions 96 and 110, Bie I ‘of all other eukaryotes contains only one cysteine, position 96 being replaced by serine. ; an Histone 4 (MW 11,284) is highly conserved, aud its amino acid sequence is nearly the same in all plants and animals. Its estimated mutation rate is 0.26 per 100 amino acid residues per 100 million years, Like H3 it is arginine rich. The highly conserved nature of Ht can be made out from the fact that the protein from pea seedlings differs from that of bovine thymus by only two amino acid residues out of 102. The lysine/arginine ratio is 0.79. H4 has Ac-serine at the N-terminal and glycine at the C-terminal. Ser —Gly—Arg —Gly—Lys Gly —Gly—Lys—Gly—Leu—_ 10 Gly-Lys—Gly —Gly—Ala —Lys —Arg—His—Arg—Lys— 20 al Lea Tag ic —Gln—Gly—Ie th 30 ys—Pro—Ala —Ile —Arg—Arg—Leu—Ala—Ar; —Arg—Arg— 40 Giy—Gy-—Val —Lys—Arg—Ile —Ser —Gly—tew—tle. 50 | Tr-—Giu—Giu—Thr—Are—Giy —Val —Leu—Lys—Val— 60 | e—Leu—Glu—Asn—Val—Ile —Arg —Asp—Ala—Val 70 Thr—Tyr—Thr—Glu—His —Ala —Lys Val—Thr—Ala —Met—Asp—Val —Arg—Lys—Thr— 80 Lys—Arg_Glu—Gly Ane oye Val —Tyr—Ala—Leu 90 Gly—Gly ly—Atg—Thr —Leu—Tyr—Gly—Phe— 100 102 Fig. 6-20, i i Pea cena Seqvenes of calf thymus histone 4. ea s H4 is the same exi " is solecine and residue 77 seeminn The basic residues tend 2b, 3 and 4 there ne 24 19 be clustered in histones, In histones ™ " 3 termi a predomi ; C-terminals, The intermediate reo of basic residues near the N- ™, n contains inantly BY’ predominantly Scanned with CamScanneres : General Account chromoso™ 47 cand acidic amino acids, ho erminal end. found f jstones are not found in association with the DN, nes moulds and fungi there are indications ra nina as nt, although the evidenes is of the negative type. Voliex and ssi appeat to have only a partial complement of histones, while in Bacpymena and Stylonychia all the normal histones of higher eukary- ‘peat to be present. In the mammalian viruses SV40 and polyoma tes 2b, 3 and 4 are found in the nucleoprotein complex. In Hi the dominant basic region is at pistones 2a runetions of histones. In general, histones tend to depress genetic activity. Since the histones have almost identical amino acid compositions in different anisms itis unlikely that this repression of genetic activity is selective, fiatoaes probably mask DNA in a non-specific manner. Histones also appear to have a structural role in packing of DNA molecules so as to render them more compact. In human chromosomes the packing ratio of DNA is about 10%. The ratio, however, varies during the cell cycle. Chromosomes are tightly packed greatly during ‘netaphase and highly dispersed in interphase. Synthesis of histones. The bulk of the histones are synthesized during the S phase of the cell cycle. At least 20,000 histone molecules per minute are synthesized in the cell during this phase. A small amount of histone synthesis also takes place at other stages of the cycle. The eukaryote DNA contains repetitive genes coding for histones. Histones are translated from 7-12§ mRNA. In Drosophila and mammals there are 50-100 copies of histone genes. In the sea urchins Lytechinus pictus and Psammechinus milaris there are 400 and 1,200 copies, respectively. Inthe latter urchin 0.5% to 0.8% of the genome is required to encode the histone genes and their spacer DNA. During interphase of the cell oe histone synthesis takee place simultaneously with DNA synthesis. ie been suggested that as the DNA is replicated histones bind to it “press any unwanted RNA synthesis. Nonhistone Chromosomal (NHC) Proteins Nonhistone ch; romoso} i i "egulatory functions mal proteins’ have structural, enzymic and £1 electrophoresis rot the chromatin complex. SDS polyacrylamide Peptide chains), depen: resolved 20-115 NHC proteins (individual poly- Glasses of ty C prenaine on the source of chromatin. There are five NAC proteins ‘dige Al, A2, B,C and D, each with 1-7 subfractions.\ acid i istones ee i several respects from histone proteins. “(NC Protein sic proteins while many of the NHC proteins are | 8Saclass probably overlap but are not identical Scanned with CamScannerUg Call Bio witl mn Thee Clases of acidic nuclear proteins and nuclear phospoproty, i) The asic-residues ratio of NHC proteins is 1.2 to 1.6. ), ~21,500, ee weight of histones ranges from ~11,0%9,, » fone ino ¢ individual polypeptide chains of NHC proteins ra,,° ,000 to several hundred thousand daltons. ee _. _ iii) In contrast to histones, NHC proteins show variation in structy in different species and even tissues. Histones have almost identi cd amino acid compositions in different organisms. On the other hang different organisms differ greatly in their nonhistone proteins. (iv) The bulk of histones are synthesized during the S period, whi, NHC proteins are synthesized throughout the cell cycle. (v) In general histones tend to depress genetic activity, while Nuc proteins stimulate genetic activity. Functions of NHC proteins. NHC proteins have enzymic, structur| and regulatory functions, Many enzme activities are associated with chromatin. Enzymes of chromosomal metabolism like nucleic acid polymerases, nucleases and enzymes of histone metabolism are integral components of chromatin, Several proteins affecting DNA conformation, e.g. proteins that stabilize ssDNA and superhelix unwinding proteins, have been isolated. It has been suggested that NHC proteins play a role in meiotic chromosome pairing. The synaptonemal complex of meiosis is probably made up of NHC proteins. NHC proteins are considered to be positive regulators of gent expression and stimulate genetic activity. They havea role in the derepression of the repressor activity of histones. Regions of DNA masked by histones are reactivated by NHC proteins (See : ‘Regulation of gene activity in eukaryotes’). NHC proteins are believed to play an important role in the inter’ action of steroid hormones with target cell nuclei. This interaction $ thought to lead to specific gene action. O’Malley and his coworkes have described the hormonal regulation of gene activity in cells of the hen’s oviduct (Fig. 6.21). Here the female hormones estrogen ® Progesterone trigger the synthesis of specific proteins ovalbumin 3 avidin, respectively. The mRNAs for ovalbumin increase by 40% wit two minutes of administration of estrogen. The hormones are a molecules which diffuse into and out of all cells in the body. They bee ever, affect only those cells (target cells) which contain specific 10°44 oteing, The hormone molecules bind to the receptor proteins BNA! pes eceptor complex. This diffuses into the nucleus, » and initiates specific mRNA transcription. Scanned with CamScanner(hormone) © Cell from hen's ovidvet Ribosomes Ovalbumin “1 Ovalbumin we Receptor protein | ooo © resesierone “ {hormone} Hormone receptor complex 88 ‘emcee Q 7 oa © Fi. 621, A Hota Rtulation of . B DRMoereceptor eeUe activity by hormones in the hen's ovi © ane cribes gomPlex binds to DNA. e hen's oviduct. B BNA anaes oeNA for ovalbumin, °F Seromosome. fi », NA trance teceptor "coeff forms tran mes, & hormone-receptor complex. a RRNA for aniignt™ the nucleus and binds to DNA. in avidin on the ribosome, Scanned with CamScanner120 : Cell Biology Ih i bands erosepi polytene chromosomes there is pulling of specie and ‘sponse to the hormone ecdysone. The bands become diffuse aa are the sites of new RNA synthesis. There is 100% increase jp IC proteins at the puff locus in the active state. INTERCHROMOSOMAL CONNECTIVES Recently evidence have been put forward indicating that nonhomo. terconnected by very thin logous chromosomes within apucleus are in under the electron micro. DNA fibres which are generally visible only scope. These interchromosomal connectives are supposed to be structural ranches of chromosomal DNA. (The connectives are different from the linear extensions of ‘chromosomal DNA described earlier by DuPraw) + Schneider (1972) has shown that in the opossum the connectives are the sites of RNA synthesis, Du Praw (1972) states that the DNA ofthe connectives would not generate gene maps if they are considered to be composed of highly repetitive chromosomal satellite DNA, and are hence genetically inert. Lampert (1971) has clearly demonstrated interchromo- Somal connectives in electron micrographs of some human cancer cells. ; Fig. 6.22. Diagram of | interchromosomal connective. \ CONCLUSION It will be now pertinent to consider the questions raised es * beginning of this chapter. Scanned with CamScannermosomes : General Acconnt chro 1a (1) Does the chromosome contain a single strand or multiple stands oN the chromatin fibre is treated with When the Treated with tryps; ae e,a central axis remains, This axis is sonitee i daestng with DNase and stains with uranyl acetate. From this facts it has ‘been concluded that the chromatin Fibre consists of a DNA double helix to which is attached protein. DuPraw concluded that a chromatid contains qne DNA double helix. In other words his model of the chromosome jsasingle stranded model. The nucleosome concept also favours the single-strand model. 0 . (2) Isthe DNA present as a continuous strand fromone end of the chromosome to the other, or is it interrupted by ‘linkers’ ? Ifthe DNA molecule is not a continuous thread does it consist of aseries of DNA molecules joined end to end by non-DNA ‘linkers’ 2 On the basis of data on DNA content of chromatids it has been estimated that the largest human chromosome would contain a DNA helix 7.3em Jong, and the smallest chromosome a DNA molecule 1.4 cm long (Du- Praw, 1961). By using autoradiographic methods Sasaki and Norman (1966) have demonstrated DNA molecules 1 to 2.2 cm long in human Does the DNA thread consist of a series of DNA molecules joined by protein ‘linkers’? If so, it should be broken up into many segments by protein digesting enzymes. Treatment of DNA filaments by pronase failed to break up the filaments (Sasaki and Norman, 1966). The authors therefore. conclude that if any linkers are present they are resistant to pronase. The work cited above indicates that the DNA is present asa _ Continuous thread in each chromatid. On the other hand it has been Shown that chromosomal DNA may consist of a series of segments or replicons (c. g. Taylor, 1961). Also, under certain conditions the DNA poh into segments 100 microns in length. In spite of this, how- subvaite 7 no definite evidence of non-DNA linkers between DNA inp vp qdications are that the mitotic chromosome is formed by oi iy on ., mri folding or coiling of single DNA molecule associated. with tees atin Associated with the DNA molecule? Pots to form myers DNA Of the chromosomes is associated with fe anmonly pa The proteins may be Protamines or, ‘omo: ar neraee Other types of proteins associated with There are stone chromosomal (NHC) proteins. vith different j ' vine 7 ccorgntPretations as to how the protein is associated smal] Bt00ve of to One interpretation in the case of prota- © DNA molecule ig occupied~by the poly- Scanned with CamScannerNonbasic amino acid wate Ati 3 A Petgpepentide chain (protamine) CQ " b yoy d c 6 9 % g 0.0) oO cof , 0 og 6 Ses D Fig. 6.23. Models of association of protein with DNA, ‘These models nowrendered unlikely because of the nucleosome concept. Scanned with CamScanner123 es : General Account Chromosor ' }A). Histones occupy both grooves of the DNA eptide chains een pa arrangment of ‘the association between Pr jecule. ¢ leohistone is shown in Fig. 6.23B. (3) The histone a pNA and uc represented as bridges across the turns of the _ exes ate also 23C). (4) According to DuPraw the DNA molecule coils (FB. ied configuration by histones and other DNA linked jg held in its nates been visualized as being wedge-shaped aie proteins. eae sites each (6. 23D). (5) The nucleosome concept is wi err of DNA-protein association. Jatest MO GiANT CHROMOSOMES, hromosomes are of two types, polytene chromosomes (salivary ff Snares) and lampbrush chromosomes. gland cl ‘ENE CHROMOSOMES(SALIVARY GLAND CHROSOMES) POLYT! Normally chromosom: isi ing interphase. Polytene are not visible during interp! tene aid are. exceptions, They were first observed by: Balbiani chromosomes ; 5 828. Salivary sland chro: Salivary gland 3B Details © Detaits mosomes of Drosophila. chromosomes from the nucleus of a male. of Fig. A, central Part, °f part of a chromosome showing bands of chromomeres. Scanned with CamScanneres Celt Biolog (1881) in the salivary glands of the midge Chironomous, and are hep, called salivary gland chromosomes. Also,all the chromosomes of a path cular type in the individual have an equal number of bands with simijj, distribution. Topographical maps of the bands and interbands have been made, parallel to genetic maps. In the four chromosomes of Drosophij, over 5,000 bands have been found (Fig. 6. 24). The differences betwee, the bands and inter-bands are shown in the following table. Table 6.4 Bands Interbands 1, Stain intensely with basic 1. Do not stain intensely, stains. | 2. Feulgen positive. 2. Feulgen negative. 3. Regions of high DNA con- 3. Regions of low DNA con centration. centration. 4. Chromonemata (unit chro- 4. Chromonemata relatively ex. matids) tightly packed by fold- tended. ing. 5. Absorb ullra-violet light at 5, Absorb little ultra-violet light. 2,600A. The work of Painter (1933)-and Bridges (1936) showed that the bands of polytene chromosomes have a close relationship to genes. b Certain genetic defects are associated with specific bands. Also muta- tions are associated with certain bands (deletions). This led to the con- cept that the bands are the sites of genes (DNA), and that the interbands are relatively inert Jinker regions. If this were so there would be only about 5,000 genes in Drosophila. This number is’ far too low. Itis now believed that the interbands also contain many genes. Recett discoveries suggest that the DNA extends continuously through the bands and interbands. This led DuPraw and Rae (1961) to suggest that # gene may overlap from band to interband, or may even lie entirely in the interband. Some bands may contain several gene loci, and # single band may appear as two after stretching. Unit chromatids. In the salivary gland chromosomes of Drosophilé DNA is concentrated in the bands, while the interbands have very littl¢ DNA. However, the fact that the interbands do eontain DNA has bec! established by quantitative staining with Feulgen stain, by flouresce™ dyes and by tritium-thymidine labelling. DNA is associated with prote (both histone and nonhistone) to form nucleoprotein fibres (100-5004) DuPraw and Rae (1966) have shown that these fibres are comparable © Scanned with CamScannerosomes : General Account carom 125 leoprotein fibres that ar , 9-500A nucl S that are found in typical j ave postulated that 1 'ypical interphasic They have p he fibres Sottespond to unit chromatids ym . i ts formed by cory unit chromatids. Each unit chromatid. consists of 4 ston! DRA omle, several centimeters long, associated with protein tie, NS lee inuous from one end of the chromosome to ; vain he other, wf congalded in the bands and relatively extended in the ine egone (puree, 1970). ine 10 nuclei interbands. Chromosome puffs The bands of polytene chromosomes become enlarged at certain nes to form swellings which have called chromosome puffs or Balbiani times rman and Bahr (1954) have studied the fine structure of the ae ‘According to them the puffs represent regions where the tightly ‘Jed chromosomal fibres open out to form many loops. Thus puffing is ono unfolding or uncoiling of individual chromomeres in a band. The due foe active genes and represent sites of RNA synthesis, pul perimental evidence for WRNA synthesis at puffs. There are several evidences to show that puffing patterns along the chromosome represent gene activity. (I) The puffs contain large amounts of RNA. The un- qulfed bands contain chiefly DNA and histone (protein). When chromo- somes are stained with tolouidine blue, RNA is stained reddish violet and DNA blue. (2) Pelling injected radioactive uridine into Chironomous larvae which were then killed and processed for autoradiography. Only the puffs were labelled with black dots, indicating RNA formation, some- times as quickly as two minutes afterwards. The rest of the chromosome and cytoplasm showed very little activity. (3) Treatment with the anti- biotic actinomysin D, which is an inhibitor of RNA synthesis, stops RNA formation. (4) When radioactive Jeucine or amino acid is injected the proteins take up no radioactive material. This leads to the conclusion that the proteins in the puff are not made in the chromosome but else- where. This is supported by the fact that inhibitors of protein synthesis like chloramphenicol and puromycin have no effect on puffing. ese Tutsi Analysis has revealed that the RNA in the puff is RNA sy ‘A (mRNA), It has been found that the puffs are active in inthe a iss but not in protein synthesis. The mRNA is synthesized Ssociates With a DNA template. It moves to the cytoplasm where it Of special wroittie Semee and acts as a template for the synthesis made in another, he mRNA of one puff varies from the mRNA _Conditic dung netamor a ig. ‘Clever has'studied changes in puffing patterns ve obaetiat Is in Chironomous. Many types of puffing patterns | (1) Some bands do not puff ‘at all. (2) Others Scanned with CamScanner126 Celt Blolyy ‘undergo puffing but there is no connection between outing and Tou, ing. (3) Puffs are present during the intermoult stage bu COME lt during metamorphosis. Thus here there is mesiatans 2 ty already present. (4) Puffing begins at the sommencent as Tout (S) Puffing begins some time after the somiencemen “ fa ting, Sings Puffing represents gene activity the conclusion is ; ; Heel Bene, become active at different stages. A tise in the level of fe e moult hormone ecdysome is concurrent with the appearance fet 0 ul, A and B, located on chroniosomes I and IV respectively. Thus eedysont acts as an inducer for gene activity (protein synthesis) in genes Aandy, Differential gene activity. Beermann (1961) has described differen. tial gene activity in Chironomous. In C. pallidivittatus four Cells near the duct of the salivary glands produce a secretion (protein). A group of bands at the tip of chromosome IV undergo puffing, showing that these bands control the secretion. In C.tentans the four cells produce a non. granular secretion and there is no puffing at the tip of chromosome ly, In the hybrid of C. pallidivittatus and C, tentans fewer granules are Pro duced and the puff is formed only at the tip of one chromosome, This is to be expected if it is assumed that differential Bene activity occurs, A gene in a specific type of cell puffs regularly, while the same gene in another type of cell does not, LAMPBRUSH CHROMOSOMES Lampbrush chromosomes are so called because they appear lke th Blass chimneys of kerosene lamps. Stu dies on these chromosomes have contributed much to our knowledge of chromosome structure and function, The first description of lampbrush chromosomes was by Flemming (1882) on amphibian oocytes, A detailed study of the lampbrush chromosomes was made by J. Ruckert (1892) 02 the oocytes of the shark. (Chaetognath), Sepia (moll#) Species of insects, sharks, a” » The lampbrush chromosomes of invertebrate ‘ose of vertebrates, although they ba the characteristic 7 ‘ance. They have been reported in pis Tecent studies are tj ts (Callan # Hoyd,1961; awa, Allfrey ana Mirsky, 1963), ese on|pewts( SIZE. , . Tn many ani than 1,000 mi Y animals ta Scanned with CamScannerGeneral Account ’ 127 ie pmosomes * es contract and are greatl th al oll further during metaphase I. In ern ea prush chromosomes may reach a | + to sh chromosomes are very clastic, id en be nem amptiyo ties before they break. upto ab TR UCTURE. During early prophase lampbrush chromosomes jn the form of a pair of homologous chromosomes having a few of contact of chiasmata (Fig. 6.26 A). Under the light micro- e each ‘chromosome is seen to consist of an axis along which is a aoe dense granules OF chromomeres. From each chromomere arises 10¥ of tera loops. (Fig. 6.26 B), 1 Chromosomal axis. Each chromosome of the pair of homolo- = ¢hromosomes consists of two chromatids, which are represented by or laments. THUS the pair of homologous chromosomes has four filaments in all. The axial filaments and the chromomeres consist of DNA. The loops represent lateral extensions of the axial filaments. At axial filaments are small swellings without loops. These the ends of th 7 are the telomeres. Each bivalent also contains a region called the centromere, Which is loop-free. 2. Chromomeres. At certain points along theii ments become tightly coiled. These points are the chromomeres. The chro- momeres are found in pairs, one chromomere for each filament. They perhaps correspond to heterochromatin (Ris, 1957). 3, Loops. Loops are of two main types, typical and special (Fig. 6.25). Most of the loops are typical. Each pical loop consists of a central axis tom which ate given off RNA firs gressively increasing lengths. This They may In some salamander am chromos ct ints: r length the axial fila- aa oop markedly thicker on one ® aymmetty eee loops have a-marked Fig. 6.25.Lateral loops. ind have granules at the end (a) Typical loop, (B) Special loop. of the fibrils, _ Each loo) ; fe. 6.5 Dy anit of aa axial fibre which is covered with a matrix pike Which breaks aa the loop is treated with deoxyribonuclease (an vor Dyas foww DNA) itis broken down, indicating that i S down RNA) ue Meated with ribonuclease (an enzyme which + fypsin-and pepsin, the matrix of the loop is Scanned with CamScannerPromoter region chains Al n Sy i ¥ SHS ES SPLZERS A P Fig. 6.26. Diagrams of the giant lamb og 00? ' Of the new! Trimet mbrush chromosomes af the develop mm 9 Ape A pair of meiotic chromosomes, joined by two chiasmata. . A part of the chromosome enlarged Jateral 100P*, . Stfetehing of the chromosoine shea oma ser ao with the axis of the chromosome. . A part of axis and a palit of loops, Note the RNA-prott ead being transcribed on the DNA thre . Diagram of a tandem series of geneas ", One gene locus enlarged, Scanned with CamScannersomes : General Account. Chromo: 5 removed. From this it can be concluded that the matrix consists of RNA and protein. Electron microscope studies by Miller and Beatty (1969) on the Jampbrush chromosomes of the oocytes of the salamander Triturus yiridescens show the presence of dense granules on the DNA axial fibre. These granules are probably large molecules of the enzyme RNA poly. merase, Which synthesizes RNA. Arising from these RNA polymerase molecules are fine fibrils of RNA-protein (ribonucleoproteins). The loop is believed to represent one long operon consisting of a series of identical copies of the same structural genes (cistrons) (Fig.6.25 Eand 6.26 F). Each gene locus codes for 45S rRNA in the | Triturus oocyte. Each locus probably produces a very long RNA mole- cule, This then interacts with protein to form riboncleoprotein particles, Between the gene loci are matrix-free regions of ‘spacer’? DNA which apparently do not produce RNA. At one end of the spacer DNA region can be seen dense granules of RNA polymerase attached to the promoter recion, The other end is the chain termination region, ~ Callan and Liyod (1960) have suggested that each pair of loops is associated with the activity of a specific gene. Each loop is supposed tocontain repeating DNA sequences (genes) arranged ina series. In other words there are several identical copies of genes in eachloop. At each chromomere is supposed to be a ‘master gene’ copy which transfers information to several ‘slave gene’ copies on the same loop (‘master and slave hypothesis’). Only the slave genes take part in RNA synthesis, but not the master gene. A high rate of synthesis is possible because of the presence of repeating similar genes. It has been proposed that the loop continually spins out from the chromomere at the thin end and rewinds at the thick end. Suggested Reading Alt tehurer, W.,G. Klobeck, W. Horz and H. G. Zachau (1978). Studies on chro- Sosa, tructure by nuclease digestion. Federation of European Biochemical peg! Spt llth meeting Copenhagen 1977, Vol. 43, Symposium Bery, RP (1968). Pergamon Press, Oxford, qT ee aa Tost. Monoge, fa oe and the synthesis of ribosomes, Nat. Cancer, 8.W. Catan, Gat, etrochromation. Science, 151,.417-425. Dang, Titruscrignee? Ls (1960). Lampbrush chromosomes of crested newts, ’tligton, C.D, vs (Laurenti), Phil. Trans, Roy. Soc., London. —243,135-219. . and Wyli Dt, C2048 Allen Midian P. (1955). Chromosome atlas of flowering plants. MB, ana Inwin, London, shaw, M, if otomes, pee ae (1971), Specific banding parterns of human chro- "Nat. Acad. Sci, U.S. A. 68, 2073-71. Scanned with CamScanner