DROSOPHILA CULTURE

Introduction

In order to incorporate D. melanogaster in the classroom, it will be necessary to maintain

cultures of flies for manipulation in crosses and as a backup for any mishaps which may occur. Culturing
is very easy and it is recommended to have students maintain their own cultures of flies. In that way,
each student or group would be directly responsible for the care and long-term maintenance of the flies,
including making large culture populations for their crosses. When directly involved, students gain
proficiency and a greater understanding of the flies requirements and behavior. The teacher should
remain as coach, not lecturer, assisting students in techniques. The instructor needs to maintain stock
cultures of all strains and mutants used by students in case the afore- mentioned unforeseeable incident
occurs and student cultures die out or become intermixed. Losing cultures is the exception rather
thanthe rule, and as long as students reculture their flies on a regular basis and no mass contamination
occurs (see pests and diseases section), flies can be maintained for decades.
REVIEW OF RELATED LETIRATURE

Structural and biochemical characterization of the P-element transposase of Drosophila

melanogaster

Alejandro Sabogal

University of California, Berkeley, 2009

Transposons are mobile DNA segments that can change genomic location within an organism, or
horizontally transfer to a new organism. They exist in all model organisms studied to date, both
prokaryotic and eukaryotic, and their mobility can cause genomic rearrangements and over time,
contribute to genome evolution. Transposition into a new genomic location can potentially change a
protein coding segment, a regulatory transcriptional sequence, or any number of direct or indirect
changes towards gene expression patterns in the cell. Although the mobile P-element is used as a
molecular tool to genetically manipulate protein coding and regulatory segments in the model system
Drosophila melanogaster, little mechanistic detail is known at the molecular level about the mechanism
of P-element transposition. The full length P-element transposon is 2.9 kb, containing a four-exon, multi-
domain protein flanked by perfect inverted repeats that are required for transposition of the element.
We have taken a biochemical and structural approach towards a molecular understanding the P-
element transposase mechanism; here we describe the mechanism of high-affinity site-specific DNA
binding by the N-terminal THAP domain through x-ray crystallography, as well as mapping of the site-
specific DNA-binding, dimerization, and GTP-binding domains onto the primary sequence of transposase
through a chimeric-GFP solubility screen, for future structural studies.

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Activation of recombinant trp by thapsigargin in Sf9 insect cells

LUIS Vaca, WILLIAM G Sinkins, YANFANG Hu, DIANA L Kunze, WILLIAM P Schilling

American Journal of Physiology-Cell Physiology 267 (5), C1501-C1505, 1994

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to
the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein
designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called
capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective
inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane
current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate
that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel
that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin,
whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store.
Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for
Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A
unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for
capacitative Ca2+ entry.

AL Mammen, JA Mahoney, A St Germain, N Badders, JP Taylor

UBE4B Is, 2011

Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues
later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a
function in the brain has been well established in vivo, and in vitro studies have shown that its activity is
essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2
alternative splice forms of UFD2a, UFD2a-7 and-7/7a, are expressed sequentially during myoblast
differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in
mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an
additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a
similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal
muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated
muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific
splicing regulatory motifs located in the 3! introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an
AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their
interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with
VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its
muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most
likely imparts divergent functions that are important for myogenisis.

Citation: Mammen AL, Mahoney JA, St. Germain A, Badders N, Taylor JP, et al.(2011) A Novel Conserved
Isoform of the Ubiquitin Ligase UFD2a/UBE4B Is Expressed Exclusively in Mature Striated Muscle Cells.
PLoS ONE 6 (12): e28861. doi: 10.1371/journal. pone. 0028861

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The statistical and molecular logic of gene expression patterns in Caenorhabditis elegans

Steven Andrew McCarroll

University of California, San Francisco, 2004

Gene regulation uses transcriptional control systems with a molecular logic we seek to understand.
Genome-scale sequence and expression data increasingly make it possible to use genomic patterns in
sequences and gene expression levels to reveal the logic of transcriptional regulation. In this
dissertation, two approaches to understanding transcriptional regulation are developed and applied.
First, we describe a novel method for identifying phylogenetic conservation in genomic transcriptional
patterns. We use this new approach to identify gene expression programs in aging, development, and
mRNA degradation that are shared by organisms as diverse as the nematode Caenorhabiditis elegans,
the fruit fly Drosophila melanogaster, the yeast Saccharomyces cerevisiae, and the human Homo
sapiens. We use this approach to search databases of gene expression patterns to identify relationships
among the physiological programs of diverse organisms. Second, we use a statistical approach,
probabilistic segmentation, to identify candidate transcriptional control sequences in the promoters of a
large gene family, the chemosensory receptor genes in C. elegans. We identify many new candidate
transcriptional control sequences and show that one of these is a novel E-box motif that confers
expression in the ADL chemosensory neurons.