Gene regulation and evolution in the chorion locus of Bombyx mori. Structural and developmental characterization of four eggshell genes and their flanking DNA regions

We report on the detailed structural and developmental characterization of four chorion genes and a truncated pseudogene located within a 9.5 X 10(3) base chromosomal segment. These genes belong to the A and B multigene families and, like previously characterized moth chorion genes, are arranged in tightly linked pairs, which are divergently transcribed (A/B.L11 and A/B.L12). On the basis of their high degree of sequence divergence, the A genes define two distinct subfamilies, while the more homologous B genes represent different copies of the same gene type. The A.L11 and B.L11 introns are much longer, in each case because of a single inserted DNA segment that is missing from A.L12 or B.L12. The 2.1 X 10(3) base insertion in A.L11 is the first retrovirus-like transposable element characterized in Bombyx mori. The very short 5' flanking sequences of A/B.L11 and A/B.L12 (277 and 276 base-pairs) are distinct as shown by hybridization but both recur in additional chorion gene pairs, forming two respective classes that are expressed during distinctly different developmental periods. The divergently transcribed genes of each pair, which border the same 5' flanking sequence, are expressed co-ordinately, during the same developmental period. Detailed comparisons of the 5' flanking regions, and of the corresponding region of the Drosophila s15-1 chorion gene, revealed numerous, very short sequence elements that are shared. One such element, T-C-A-C-G-T, is also associated with all five sequenced Drosophila chorion genes. Some elements are repeated in a dyad symmetrical pattern, i.e. are associated with each of the two genes in a pair, while others, including T-C-A-C-G-T, occur only once per 5' flanking region, and, if functionally important, would presumably act bi-directionally on both genes of the pair.     

The chorion genes of the medfly, Ceratitis capitata. II. Characterization of three novel cDNA clones obtained by differential screening of an ovarian library

We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library. Their isolation was accomplished by differential screening of the library using as probes 32P-labeled poly(A)+ mRNAs obtained from hand-staged medfly choriogenic versus prechoriogenic follicles. RNA blot hybridization analysis revealed that the genes corresponding to these clones have unique temporal profiles of mRNA accumulation, restricted to specific choriogenic stages. In addition, in vitro translation products encoded by these cDNAs approximately comigrated with polypeptides synthesized de novo in culture by choriogenic follicles. All three genes are located in regions of the medfly genome that are specifically amplified in female ovaries. DNA sequence analysis has revealed that one of these clones is derived from a homolog of the Drosophila melanogaster s38 chorion gene. It appears that, although D. melanogaster and C. capitata are separated by at least 120 million years of evolution, the mechanisms by which chorion genes are expressed and regulated during development have been well maintained. We suggest that the regulatory elements controlling the expression of sex-specific (e.g., chorion) genes may be isolated and used to construct transgenic medfly strains from which females could be eliminated by negative selection; such strains could be used as part of an effort to control this agricultural pest.     

Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network

The sequence of paired, a pair-rule gene required for segmentation in Drosophila, is presented. A search for genes with domains homologous to the paired gene was initiated and three homologues from a set of 12 were characterized with respect to temporal or spatial expression and sequence homologies. All four are transcribed in early development, one in the oocyte and during cleavage stages in the form of a gradient. In addition to the prd-specific his-pro repeat, some of the 12 genes contain M-repeats and two new types of homeo boxes not detectable by hybridization with the two known classes of homeo boxes. The observed linking of gene sets through combinations of homologies coding for protein domains is consistent with a general network concept of gene action.     

Evolution of the Autosomal Chorion Locus in Drosophila. I. General Organization of the Locus and Sequence Comparisons of Genes S15 and S19 in Evolutionarily Distant Species

We have isolated clones corresponding to the autosomal chorion locus of Drosophila melanogaster, from two distantly (D. virilis and D. grimshawi) and one closely (D. subobscura) related species. In all the species the locus is unique within the genome and encompasses the same four chorion genes and an adjacent nonchorion gene, in the same order. In all species the locus specifically amplifies in the ovary, as in D. melanogaster. We present the nucleotide sequences of DNA segments that total 8.3 kb in length and include gene s15-1 from D. subobscura, D. virilis, and D. grimshawi as well as gene s19-1 from D. subobscura and D. grimshawi. They show clearly nonuniform rates of divergence, both within and outside the limits of the genes. Highlighted by a background of extensive sequence divergence elsewhere in the extragenic region, highly conserved elements are observed in the 5' flanking DNA and might represent regulatory elements.     

The biolistic method as a tool for testing the differential activity of putative silkmoth chorion gene promoters

Bombyx mori unpaired early chorion gene copies 6F6.1,.2 and.3 are exceptions to the typical organization and distribution pattern of known early ErA/ErB, middle A/B and late HcA/HcB divergently transcribed gene pairs. Contrary to such pairs, the boundaries of the 6F6 regulatory sequences are not easily defined; moreover, they share common sequence elements with the regulatory sequences of middle and late genes. In order to perform a functional study of the tissue and temporal specificity of the 6F6 putative promoter region, we decided to apply biolistics. In the present work, use of a region from the 6F6.2 5' untranslated sequence, spanning nucleotides -138 to the cap site, gave an expected expression pattern of a lacZ reporter gene. Temporal specificity was further verified by control experiments using the cloned intergenic sequence of the late gene pair HcA/B.12, which resulted in lacZ expression in late choriogenic follicles. At present, despite the recent successful germinal transgenesis of Bombyx mori, the biolistic transient expression system seems to be the most rapid technique to pursue the functional study of the promoter region of early chorion genes, including the three unconventional early 6F6 genes.     

Time ordering of gene coexpression

Temporal microarray gene expression profiles allow characterization of gene function through time dynamics of gene coexpression within the same genetic pathway. In this paper, we define and estimate a global time shift characteristic for each gene via least squares, inferred from pairwise curve alignments. These time shift characteristics of individual genes reflect a time ordering that is derived from ob- served temporal gene expression profiles. Once these time shift characteristics are obtained for each gene, they can be entered into further analyses, such as clustering. We illustrate the proposed methodology using Drosophila embryonic development and yeast cell-cycle gene expression profiles, as well as simulations. Feasibility is demonstrated through the successful recovery of time ordering. Estimated time shifts for Drosophila maternal and zygotic genes provide excellent discrimination between these two categories and confirm known genetic pathways through the time order of gene expression. The application to yeast cell-cycle data establishes a natural time order of genes that is in line with cell-cycle phases. The method does not require periodicity of gene expression profiles. Asymptotic justifications are also provided.     

The alpha-amylase gene in Drosophila melanogaster: nucleotide sequence, gene structure and expression motifs.

We present the complete nucleotide sequence of a Drosophila alpha-amylase gene and its flanking regions, as determined by cDNA and genomic sequence analysis. This gene, unlike its mammalian counterparts, contains no introns. Nevertheless the insect and mammalian genes share extensive nucleotide similarity and the insect protein contains the four amino acid sequence blocks common to all alpha-amylases. In Drosophila melanogaster, there are two closely-linked copies of the alpha-amylase gene and they are divergently transcribed. In the 5'-regions of the two gene-copies we find high sequence divergence, yet the typical eukaryotic gene expression motifs have been maintained. The 5'-terminus of the alpha-amylase mRNA, as determined by primer extension analysis, maps to a characteristic Drosophila sequence motif. Additional conserved elements upstream of both genes may also be involved in amylase gene expression which is known to be under complex controls that include glucose repression.     

The Drosophila RBP-J kappa gene encodes the binding protein for the immunoglobulin J kappa recombination signal sequence.

We previously isolated a cDNA encoding the 60-kDa murine protein (RBP-J kappa protein) that specifically binds to the immunoglobulin J kappa recombination signal sequence. The RBP-J kappa gene is highly conserved in a wide variety of organisms including man, Xenopus, Drosophila, and yeast. We have isolated and characterized the Drosophila homologue of the RBP-J kappa gene. The Drosophila RBP-J kappa gene was mapped to the polytene region 35BC of chromosome 2. The nucleotide sequence of this gene indicates that it is not one of the known genes located in the 35 BC region. The nucleotide and amino acid sequences of the Drosophila and mouse RBP-J kappa genes are 60 and 75% homologous, respectively. The central 248-residue regions of RBP-J kappa proteins of the two species are 93% homologous and include the 40-residue integrase motif. The Drosophila RBP-J kappa protein expressed in COS cells bound to the J kappa recognition sequence with the same specificity as the murine counterpart. These results suggest that Drosophila may have a site-specific recombination system which utilizes the immunoglobulin recombination signal sequence. Implications for evolution of immunoglobulin gene rearrangement were also discussed.     

Chorion cDNA clones of D. melanogaster and their use in studies of sequence homology and chromosomal location of chorion genes

Clones corresponding to two distinct A1 and A2 chorion genes have been isolated from a cDNA library in Drosophila melanogaster and characterized by hybrid-selected translation and blotting-hybridization analysis. These sequences detectably cross hybridize, thus indicating that at least some chorion genes in the fruit fly are homologous. According to in situ hybridization results, the A1 and A2 genes are not linked (mapping in regions 66D 10-12 and 54C-D of the third and second chromosomes, respectively). In conjunction with other evidence, these results suggest that in Drosophila, clustering of chorion genes may be limited to genes which are expressed in parallel during development.     

Genomic analysis of synaptotagmin genes.

I used TBLASTn to probe DNA sequence databases with a consensus peptide sequence corresponding to the most highly conserved region of the rodent synaptotagmin (Syt) gene family, which is within the C2B domain. I found human homologues for all known rodent genes, and found six further human genomic loci which encode potential family members. I found eight potential family members in Caenorhabditis elegans, six in Drosophila melanogaster, and four in Arabidopsis thaliana. The C. elegans Syt1 homologue uniquely encodes two alternative C2B exons, one or the other of which is expressed at a time. Comparison of the genomic structures of the Syt genes makes clear the different phylogenies of the different subgroups. Knowledge of the genomic structures will aid the systematic investigation of alternative splicing in Syt genes.     

In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors

This is the first attempt to identify regulatory elements that are involved in early choriogenesis of the silkworm Bombyx mori. A new cis element in the promoter region of five early chorion genes was identified. The consensus sequence of this element matches the consensus of the C/EBP DNA binding site. Moreover, this sequence interacts with a 70 kD protein (pX2) present in follicular nuclear extracts and complex formation exhibits early developmental specificity. There is strong evidence that this factor belongs to the C/EBP family. Surprisingly, the same protein binds with the same developmental specificity to a similar sequence of a late chorion gene promoter, which has been previously defined as the binding site for a putative late specific factor, BCFII. The possibility that pX2 and BCFII are isoforms or modifications of the same protein factor, which is presumably able to bind to the highly similar sequence elements of both early and late genes, is discussed. A hypothesis involving protein-protein interactions between C/EBP (pX2/BCFII) and GATA during choriogenesis is presented to explain the temporal specificity of chorion genes.