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This glossary is designed to help the reader with the ter- Term Section
minology of molecular biology. Each year, the glossary c-rel X
will be expanded to include new terms introduced in the Calcium phosphate VI
Education Program. The basic terminology of molecular CAN X
biology is also included. The glossary is divided into sev- CAT V
eral general sections. A cross-reference guide is included cDNA II
to direct readers to the terms they are interested in. The cDNA blunting IX
hope is that this addition to the Education Program will cDNA library preparation IX
further the understanding of those who are less familiar cdk V
with the discipline of molecular biology. cdkI V
CFB IX
CROSS-REFERENCE GUIDE Chimeraplasty VIII
Chitosan-DNA VIII
Term Section Chloramphenicol transferase V
Actinomycin D pulse experiments V Chromatography, gel filtration IV
Adeno-associated viral vectors VIII Chromatography, ion exchange IV
Adenoviral vectors VIII Chromatography, hydrophobic IV
ALK X Chromatography, affinity IV
Allele-specific hybridization XI Chromatography, high performance
Allele-specific PCR IV liquid (HPLC) IV
AML-1 X Cis-acting factors V
Amphotropic virus VIII Codon II
Anaplastic lymphoma kinase X Color complementation assay XI
Antisense oligonucleotides VIII Comparative gene hybridization IV
Basic helix-loop-helix proteins V Competitive oligonucleotide hybridization XI
Bcl-1 X Concatamerization VI
Bcl-2 X Cyclin-dependent kinase V
Bcl-3 X Contig VII
Bcl-6 X Cosmid II
 galactosidase V CpG nucleotide II
Branched chain DNA signal Cyclins V
amplification assay II DEAE dextran VI
c-abl X DEK X
c-fos X Dideoxynucleotide (ddN) chain
c-jun X termination sequencing IV
c-myb X Directional cloning IX
c-myc X DNA (deoxyribonucleic acid) II
c-ras X DNA methylases III
DNA polymerase III
DNAse footprinting IV
* University of Washington School of Medicine, Division of He-
matology, Box 357710, Seattle WA 98195-7710 DNAse hypersensitivity site mapping IV
DNA (deoxyribonucleic acid) The polymer constructed ORF (open reading frame) The term given to any stretch
of successive nucleotides linked by phosphodiester bonds. of a chromosome that could encode a polypeptide se-
Some 3 x 109 nucleotides are contained in the human quence, i.e., the region between a methionine codon
haploid genome. During interphase, DNA exists in a (ATG) that could serve to initiate proteins translation,
nucleoprotein complex containing roughly equal amounts and the inframe stop codon downstream of it. Several
of histones and DNA, which interacts with nuclear ma- features of the ORF can be used to judge whether it actu-
trix proteins. This complex is folded into a basic struc- ally encodes an expressed protein, including its length,
ture termed a nucleosome containing approximately 150 the presence of a “Kozak” sequence upstream of the ATG
base pairs. From this highly ordered structure, DNA rep- (implying a ribosome might actually bind there and ini-
lication requires a complex process of nicking, unfold- tiate protein translation), whether the ORF exists within
ing, replication, and splicing. In contrast, gene transcrip- the coding region of another gene, the presence of exon/
tion requires nucleosomal re-organization such that sites intron boundary sequences and their splicing signals, and
critical for the binding of transcriptional machinery re- the presence of upstream sequences that could regulate
side at internucleosomal junctions. expression of the putative gene.
Branched chain DNA (b-DNA) A method that exploits Plasmids Autonomously replicating circular DNA that
the formation of branched DNA to provide a sensitive are passed epigenetically between bacteria or yeast. In
and specific assay for viral RNA or DNA. The assay is order to propagate, plasmids must contain an origin of
performed in a microtiter format, in which partially ho- replication. Naturally occurring plasmids transfer genetic
mologous oligodeoxynucleotides bind to target to create information between hosts; of these, the genes encoding
a branched DNA. Enzyme-labeled probes are then bound resistance to a number of antibiotics are the most impor-
to the branched DNA, and light output from a chemilu- tant clinically. The essential components of plasmids are
minescence substrate is directly proportional to the used by investigators to introduce genes into bacteria and
amount of starting target RNA. Standards provide quan- yeast and to generate large amounts of DNA for manipu-
titation. The assay displays a 4 log dynamic range of de- lation.
tection, with greater sensitivity to changes in viral load
than RT-PCR-based assays. It has been employed to quan- Phage A virus of bacteria, phage such as lambda have
titate levels of HIV, HCV, and HBV. been used to introduce foreign DNA into bacteria. Be-
Telomere A repeating structure found at the end of chro- Ribonuclease These enzymes degrade RNA and exist as
mosomes, serving to prevent recombination with free- either exonucleases or endonucleases. The three most
ended DNA. Telomeres of sufficient length are required commonly used ribonucleases are termed RNase A,
to maintain genetic integrity, and they are maintained by RNase T1, and RNAse H (which degrades duplex RNA
telomerase. or the RNA portion of DNA•RNA hybrids).
CpG This under-represented (i.e. < 1/16 frequency) di- Ribozyme A complex RNA molecule that possesses
nucleotide pair is a “hotspot” for point mutation. CpG endoribonuclease activity. A highly specific RNA se-
dinucleotides are often methylated on cytosine. Should quence can generate secondary structure by virtue of
Me-C undergo spontaneous deamination, uracil arises, intrachain base pairing. “Hairpin loops” and “hammer
which is then repaired by cellular surveillance mecha- head” structures serve as examples of such phenomena.
nisms and altered to thymidine. The net result is a C to T When the proper secondary structure forms, such RNA
mutation. molecules can bind a second RNA molecule (e.g. an
mRNA) at a specific location (dependent on an approxi-
III. ENZYMES OF RECOMBINANT DNA TECHNOLOGY mately 20-nucleotide recognition sequence) and cleave
at a specific GUX triplet (where X = C, A, or U). These
A. Nucleases molecules will likely find widespread use as tools for
specific gene regulation or as antiviral agents but are evo-
A number of common tools of recombinant DNA tech- lutionarily related to RNA splicing, which in its simplest
nology have been developed from the study of the basic form is autocatalytic.
enzymology of bacteria and bacteriophage. For example,
most unicellular organisms have defense systems to pro- B. Polymerases
tect against the invasion of foreign DNA. Usually, they
specifically methylate their own DNA and then express DNA polymerase The enzyme that synthesizes DNA
restriction endonucleases to degrade any DNA not ap- from a DNA template. The intact enzyme purified from
propriately modified. From such systems come very use- bacteria (termed the holoenzyme) has both synthetic and
ful tools. Today, most restriction endonucleases (and most editing functions. The editing function results from nu-
other enzymes of commercial use) are highly purified clease activity.
from either natural or recombinant sources and are highly
reliable. Using these tools, the manipulation of DNA and Klenow fragment A modified version of bacterial DNA
RNA has become routine practice in multiple disciplines polymerase that has been modified so that only the poly-
of science. merase function remains; the 5'➝3' exonuclease activity
has been eliminated.
Exonuclease An enzyme that digests nucleic acids start-
ing from the 5' or 3' terminus and extending inward. Thermostabile polymerases The prototype polymerase,
Taq, and newer versions such as Vent and Tth polymerase
Endonuclease An enzyme that digests nucleic acids from are derived from microorganisms that normally reside at
within the sequence. Usually, specific sequences are rec- high temperature. Consequently, their DNA polymerase
ognized at the site where digestion begins. enzymes are quite stable to heat denaturation, making
them ideal enzymes for use in the polymerase chain re-
action.
Nested PCR By using an independent set of PCR prim- Western blotting This technique is designed to detect
ers located within the sequence amplified by the primary specific protein present in a heterogenous sample. Pro-
set, the specificity of a PCR reaction can be greatly en- teins are denatured and size-fractionated by polyacryla-
hanced. In Figure 1, should the first PCR reaction yield mide gel electrophoresis, transferred to nitrocellulose or
a product of 600 nucleotides, a second PCR reaction us- other synthetic membranes, and then probed with an an-
ing the first product as template and a different set of tibody to the protein of interest. The immune complexes
primers will produce a smaller, “nested” PCR product, present on the blot are then detected using a labeled sec-
the presence of which acts to confirm the identity of the ond antibody (for example, a 125I-labeled or biotinylated
primary product. goat anti-rabbit IgG). As the original gel electrophoresis
was done under denaturing and reducing conditions, the
Real-time automated PCR During PCR, a fluorogenic precise size of the target protein can be determined.
probe, consisting of an oligodeoxynucleotide with both
reporter and quencher dyes attached, anneals between the Southwestern blotting This technique is designed to
two standard PCR primers. When the probe is cleaved detect specific DNA-binding proteins. Like the Western
during the next PCR cycle, the reporter is separated from blot, proteins are size-fractionated and transferred to ni-
the quencher so that the fluorescence at the end of PCR trocellulose. The probe in this case, however, is a double-
is a direct measure of the amplicons generated through- stranded labeled DNA that contains a putative protein-
out the reaction. Such a system is amenable to automa- binding site. Should the DNA probe hybridize to a spe-
tion and gives precise quantitative information. cific protein on the blot, that protein can be subsequently
identified by autoradiography. This technique often suf-
Allele-specific PCR By using generic PCR primers flank- fers from nonspecificity, so that a number of critical con-
ing the immunoglobulin or T cell receptor genes, the pre- trols must be included in the experiment for the results to
cise rearranged gene characteristic of a B or T cell neo- be considered rigorous.
plasm can be amplified and sequenced. Once so obtained,
new PCR primers can then be designed that are unique In situ hybridization This technique is designed to de-
to the patient’s tumor. Such allele-specific PCR can then tect specific RNA present in histological samples. Tissue
be used to detect blood cell contamination by tumor and is prepared with particular care not to degrade RNA. The
to detect minimal residual disease following therapy. cells are fixed on a microscope slide, allowed to hybrid-
ize to probe, and then washed and overlaid with photo-
Southern blotting This technique is used to detect spe- graphic emulsion. Following exposure for one to four
cific sequences within mixtures of DNA. DNA is size- weeks, the emulsion is developed and silver grains over-
fractionated by gel electrophoresis and then transferred lying cells that contain specific RNA are detected. The
by capillary action to nitrocellulose or another suitable most useful probes for this purpose are metabolically 35S-
synthetic membrane. Following blocking of nonspecific labeled riboprobes generated by in vitro transcription of
binding sites, the nitrocellulose replica of the original gel a cDNA using viral RNA polymerase. These probes give
electrophoresis experiment is then allowed to hybridize the lowest background and are preferable to using termi-
with a cDNA or oligonucleotide probe representing the nal deoxynucleotidyl transferase or alternative methods
specific DNA sequence of interest. Should specific DNA using 32P as an isotope.
be present on the blot, it will combine with the labeled
probe and be detectable by autoradiography. By co-elec- FISH (fluorescence in situ hybridization) A general
trophoresing DNA fragments of known molecular weight, method to assign chromosomal location, gene copy num-
the size(s) of the hybridizing band(s) can then be deter- ber (both increased and decreased), or chromosomal re-
mined. For gene rearrangement studies, Southern blot- arrangements. Biotin-containing nucleotides are incor-
ting is capable of detecting clonal populations that repre- porated into specific cDNA probes by nick-translation.
sent approximately 1% of the total cellular sample. Alternatively, digoxigenin or fluorescent dyes can be in-
Enhancer An enhancer is a segment of DNA that lies Nucleosomes When linear, the length of a specific chro-
either upstream, within, or downstream of a structural mosome is many orders of magnitude greater than the
gene that serves to increase transcription initiation from diameter of the nucleus. Therefore, a mechanism must
that gene. A classical enhancer element can operate in exist for folding DNA into a compact form in the inter-
Protein translation This term is applied to the assembly Helix-turn-helix This family of transcriptionally active
of a polypeptide sequence from mRNA. proteins depends on the helix-turn-helix motif for dimer-
ization. Examples include the homeodomain genes such
KOZAK sequence This five-nucleotide sequence resides as the Hox family.
just prior to the initiation codon and is thought to repre-
sent a ribosomal-binding site. The most consistent posi- Master switch genes These polypeptide products are
tion is located three nucleotides upstream from the ini- thought to regulate a whole family of genes and result in
tiation ATG and is almost always an adenine nucleotide. a cell undergoing a new program of differentiation. An
When multiple potential initiation codons are present in example of such a system is Myo D, in which activation
an open reading frame, the ATG codon, which contains a is thought to lead to differentiation along the muscle cell
strong consensus KOZAK sequence, is likely the true lineage.
initiation codon.
Zinc finger domain proteins The presence of conserved
Initiation codon The ATG triplet is used to begin histidine and cysteine residues allows chelation of a zinc
polypeptide synthesis. This is usually the first ATG codon, atom and results in the formation of a loop structure called
located approximately 30 nucleotides downstream of the the zinc finger domain. This feature is present in a large
site of transcription initiation (cap site). However, the family of transcriptionally active proteins such as the ste-
context in which the ATG resides is also important (see roid hormone receptors.
KOZAK sequence).
Actinomycin D pulse experiments The application of Cdk (cyclin-dependent kinase) A related group of cel-
actinomycin D to actively metabolizing cells results in lular kinases, present in virtually all cells, that are regu-
the cessation of new RNA transcription. Consequently, lated both positively and negatively by specific phospho-
serial determinations of specific RNA levels will allow rylation events and negatively by association with other
one to calculate the mRNA half life. Should this vary proteins, and are dependent on cyclins, present only dur-
between control and stimulated conditions, evidence is ing certain phases of the cell cycle (cdk1-activated dur-
garnered that a gene of interest is regulated at the level of ing G2/M phase, cdk2-G1/S phases, cdk4-G1/S phases,
mRNA stability. cdk6-G1 phase, cdk7-throughout the cell cycle).
Reporter genes In order to determine how a gene pro- CdkI (cdk inhibitors) Proteins that inhibit the cdks by
moter or enhancer works in vitro, that genetic element is stoicheometric combination, arresting cells in G1 phase,
often linked to a gene for which a simple assay is readily and include p27, p21 and the p16 Ink 4A family of pro-
available and whose regulation is not affected by post- teins. The latter are implicated as tumor suppressor genes,
transcriptional processes. Such reporter genes include as their deficiency in mice leads to rapid cellular prolif-
chloramphenicol acetyl transferase,  galactosidase, and eration and a high rate of spontaneous tumor develop-
firefly luciferase. The first is the most commonly used re- ment. Moreover, deficiency of p16 family members has
porter; however, more recent studies have emphasized the been associated with numerous types of human tumors,
use of the latter two reporters, as these are more sensitive to including a fraction of cases of B cell ALL and T cell
minimal changes in promoter or enhancer activity. leukemia.
CAT (chloramphenicol acetyl transferase) The bacte- VI. EXPRESSION OF RECOMBINANT PROTEINS
rial gene for chloramphenicol resistance, chlorampheni-
col acetyl transferase (CAT) is commonly used as a re- In order to exploit the techniques of recombinant DNA
porter gene for investigating physiologic gene regulation. research, one must possess a system to manufacture the
The assay depends on the ability of transfected cellular protein of interest. After identifying the gene encoding
cytoplasm to convert 14C chloramphenicol to its acety- the protein and obtaining a cDNA representation of it
lated form in the presence of acetyl CoA. The acetylated (“cloning”), the cDNA must be placed in a vector ca-
forms are separated from the 14C substrate using thin pable of driving high levels of RNA transcription in a
layer chromatography. host system capable of translating and appropriately
modifying the polypeptide to produce fully functional
 galactosidase The presence of  galactosidase activ- protein. And just like obtaining a protein of interest from
ity in the cytoplasm of transfected cells can be readily natural sources, one must purify protein from the final
detected by its ability to convert a colorless substrate to a expression system. Because of the nature of the highly
blue-colored product. This is usually assayed using a fluo- engineered systems and high levels of expression, this
rimeter. latter task is usually considerably easier using recombi-
nant methods than from natural sources. The methods
Luciferase This gene, which is the most recent reporter used to generate expression vectors are described in Sec-
gene to be used, has gained increasing acceptance be- tion IV, but the methods to purify proteins are discussed
cause of its ease of assay and extreme sensitivity. The in only a rudimentary way and are beyond the scope of
assay is based on the ability of the protein to undergo this glossary.
chemiluminescence and transmit light, detected with a
luminometer. Expression vector A plasmid that contains all of the el-
ements necessary to express an inserted cDNA in the host
Cyclins A group of proteins that vary in expression of interest. For a mammalian cell host, such a vector typi-
throughout the cell cycle. Once a threshold level is at- cally contains a powerful promoter coupled to an en-
tained, interaction with specific cellular kinases results hancer, a cloning site, and a polyadenylation signal. In
in phosphorylation of critical components of the mitotic addition, several expression vectors also contain a select-
YAC (yeast artificial chromosome) A yeast artificial Ecotropic vectors Many retroviruses are host cell spe-
chromosome (YAC) utilizes centromeric and telomeric cific, i.e. they will only infect a specific species of cells.
elements from yeast chromosomes to construct genetic An example is the widely used Maloney virus, and its
elements that can be propagated in yeast and transferred basis lies in the species-specific expression of the viral
into mammalian cells. Such vehicles allow the introduc- cell surface receptor.
tion of up to 200 kb or more of genetic material into the
host cells. YACs are now being used to study the physi- Ecotropic viruses Murine retroviruses that contain coat
ologic regulation of large genetic loci such as the -globin proteins that can only bind to murine cellular receptors.
region of chromosome 11.
Amphotropic viruses Retroviruses whose coat proteins
Contig The jargon term used to describe the assembly of bind to a receptor found throughout multiple species,
clones necessary to include all of the DNA in a specific usually including man, making these vectors suitable for
stretch of chromosome. Such maps are usually assembled human use. Problems related to the level of receptor ex-
from overlapping YAC (yeast artificial chromosome) or pression on cells of hematologic interest (e.g. stem cells)
BAC (bacterial artificial chromosome) clones. Once the remain for amphotropic viruses.
“genome project” is complete, it will consist of 24 (very
large) contigs (22 autosomal, an X and a Y). Pseudotyped virus These take advantage of the power-
ful expression levels obtainable by murine retroviral back-
Transposon Naturally occurring genetic elements that bones, yet are packaged in an envelope that allows dock-
are naturally easily removed and inserted into the genome, ing and uptake by human target cells. An example is the
allowing for the recombination of genetic segments, giv- popular MFG vector that utilizes a murine leukemia
ing rise to genetic diversity. These same elements can be retroviral backbone and an amphotropic packaging cell
utilized for gene therapy. line to produce infectious particles.
VIII. GENE THERAPY Episomal Episomal refers to gene therapy vectors that
remain free in the target cell without being taken into the
Gene therapy takes many forms. To treat malignancy, it host genome.
may involve the insertion of an adjuvant substance (such
as GM-CSF) into tumor cells to generate a tumor vac- Positional variegation Refers to the observation that the
cine, transfer of a gene that renders tumor cells suscep- site of vector integration into the genome often results in
tible to eradication with an antitumor agent (e.g., herpes variable levels of gene expression.
thymidine kinase), or insertion of a gene that makes by-
stander cells resistant to the effects of chemotherapy (e.g., Chimeraplasty A technique of gene therapy dependent
MDR). For gene deficiencies, insertion of the wild type on construction of a DNA:RNA oligonucleotide hybrid
allele is the therapeutic goal. Obtaining cDNA for de- that once introduced into a cell relies upon DNA repair