Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant

Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu ¹ females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes was also studied and the function of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.     

Extent of polyteny in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster

In the polytene nuclei of germ-line cells (ovarian pseudonurse cells) of Drosophila melanogaster females mutant for otu ¹¹ (ovarian tumor), the pericentric heterochromatin is much more abundant than in somatic salivary gland cells. This is due to the degree of heterochromatin compaction (and consequently the level of underreplication) being lower in the nurse cells than in the salivary gland cells. The lower level of compaction probably results in a very low degree of position effect gene inactivation in the ovarian nurse cells.     

Identification and genetic localization of mRNAs from ovarian follicle cells of Drosophila melanogaster.

RNA synthesis in ovarian follicles of Drosophila melanogaster was studied by methods which eliminate experimentally induced alterations in gene expression. Gel electrophoresis of follicular RNA, labeled after injection of precursors into females, revealed qualitative and quantitative differences in synthesis during the course of oogenesis. A highly heterogeneous group of poly(A)-containing RNAs is produced during much of the course of follicular development. However, post-vitellogenic stages synthesize a small number of stage-specific poly(A)-containing RNAs. During this period, RNA synthesis is known to take place primarily in the follicle cells, which are engaged in the production of the endochorion and exochorion. Two intense bands of nonmitochondrial poly(A)⁺ RNA are labeled between stage 11 and early stage 13. The synthesis of a more heterogeneous group of very small poly(A)-containing RNAs characterizes the last part of oogenesis, stages 13 and 14. Evidence is presented to show that these RNAs are specifically localized in the follicle cells of the egg chamber. We propose that they represent mRNAs for chorion proteins. In situ hybridization of preparations of late stage poly(A)-containing RNA to salivary gland chromosomes revealed two major sites of complementarity, 7E11 and 12E, as well as several minor sites. Experiments in which RNAs were separated on gels prior to hybridization in situ suggested that both the major stage 12-specific RNA bands contained molecules which were complementary to DNA in the 7E11 region. It is particularly interesting that this site is within a small chromosomal interval known to contain the gene ocelliless. Females homozygous for ocelliless have been shown to produce structurally abnormal chorions (Johnson and King, 1974).     

Identification of methylated sequences in genomic DNA of adult Drosophila melanogaster

The genome of Drosophila melanogaster contains methylated cytosines. Recent studies indicate that DNA methylation in the fruit fly depends on one DNA methyltransferase, dDNMT2. No obvious phenotype is associated with the downregulation of this DNA methyltransferase. Thus, identifying the target sequences methylated by dDNMT2 may constitute the first step towards understanding the biological functions of this enzyme. We used anti-5-methylcytosine antibodies as affinity column to identify the methylated sequences in the genome of adult flies. Our analysis demonstrates that components of retrotransposons and repetitive DNA sequences are putative substrates for dDNMT2. The methylation status of DNA encoding Gag, a protein involved in delivering the transposition template to its DNA target, was confirmed by sodium bisulfite sequencing.     

Evolutionary rearrangement of the amylase genomic regions between Drosophila melanogaster and Drosophila pseudoobscura

Two Drosophila pseudoobscura genomic clones have sequence similarity to the Drosophila melanogaster amylase region that maps to the 53CD region on the D. melanogaster cytogenetic map. The two clones with similarity to amylase map to sections 73A and 78C of the D. pseudoobscura third chromosome cytogenetic map. The complete sequences of both the 73A and 78C regions were compared to the D. melanogaster genome to determine if the coding region for amylase is present in both regions and to determine the evolutionary mechanism responsible for the observed distribution of the amylase gene or genes. The D. pseudoobscura 73A and 78C linkage groups are conserved with the D. melanogaster 41E and 53CD regions, respectively. The amylase gene, however, has not maintained its conserved linkage between the two species. These data indicate that amylase has moved via a transposition event in the D. melanogaster or D. pseudoobscura lineage. The predicted genes within the 73A and 78C regions show patterns of molecular evolution in synonymous and nonsynonymous sites that are consistent with previous studies of these two species.     

Sequence and structure of the Drosophila melanogaster ovarian tumor gene and generation of an antibody specific for the ovarian tumor protein.

Sequencing cDNA and genomic DNA from the ovarian tumor gene revealed a gene with seven introns spanning 4.5 kilobases. The proline-rich, hydrophilic otu protein is novel. An antibody prepared to a beta-gal-otu fusion protein recognized a 110-kilodalton ovarian protein which was altered in the ovaries of otu gene mutants.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Isolation and chromosomal localization of genomic DNA sequences coding for cytoplasmic superoxide dismutase from Drosophila melanogaster

A synthetic oligodeoxynucleotide 18-mer probe derived from the amino acid sequence of Drosophila melanogaster cytoplasmic superoxide dismutase (cSOD) was used to screen a D. melanogaster genomic library. One of the positive clones maps by in situ hybridization to position 68A8-9 on the left arm of polytene chromosome 3, the region to which cSOD mutants have previously been mapped genetically. Partial sequence analysis verifies the presence of cSOD-coding sequences in this clone and indicates that the intron structure of the Drosophila cSOD gene differs significantly from its human counterpart.     

Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.

Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.     

Mitochondrial localization and temporal expression of the Drosophila melanogaster DnaJ homologous tumor suppressor Tid50

The Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (tid) was identified as a homolog of all dnaJ-like genes known to date which have been well preserved in evolution. Homozygous D. melanogaster l(2)tid mutants l(2)tid¹, l(2)tid² and l(2)tid³ are characterized by neoplastic transformation of the adult integumental primordia, the imaginal discs, and the death at the time of puparium formation. The first part of this study is concerned with the identification and subcellular localization of the l(2)tid-encoded protein, Tid50. The second part examines its tissue specific expression during wild-type development and in tumorous imaginal discs. To specify the function(s) of the Tid50 protein polyclonal rabbit antibodies directed against various domains of it were generated and used for staining of Western blots and whole-mounts and paraffin sections of various tissues isolated from wild-type and mutant tumor-developing animals. To identify the mutational events leading in homozygous l(2)tid mutants to abnormal expression level of l(2)tid-encoded RNA and protein, the mutant gene was isolated from homozygous l(2)tid¹ and l(2)tid² animals and sequenced.