Selection and sequence analysis of a cDNA clone encoding a known chorion protein of the A family.

Using as criteria the size, abundance and developmental specificity of hybridizing mRNA sequences, we have selected from our chorion cDNA library a clone corresponding to a specific chorion protein, A4--cl. Comparison between the clone sequence and the largely known sequence of A4--cl validates the use of the cDNA library for sequence analysis of the chorion multigene families. The two major chorion protein families, A and B, share certain structural similarities.     

Molecular analysis of the GrB mutation in Bombyx mori through the use of a chorion cDNA library

To study the molecular basis of the Gr^B mutation, which prevents the synthesis of many stage-specific chorion proteins, a cDNA library has been constructed from wild-type chorion mRNA of Bombyx mori strain 703. By differential screening of the library with +/+ and B/B mRNAs, under appropriately stringent conditions to minimize cross hybridizations of related chorion sequences, we have selected several distinct clones corresponding to RNA sequences which are affected by the mutation (that is, are represented only in +/+ mRNA) or are unaffected (that is, are represented in both +/+ and B/B mRNAs). We show by Southern analysis that, whereas unaffected gene sequences are represented in both +/+ and B/B chromosomal DNA, affected sequences have been deleted from B/B DNA. The organization and regulation of developmental stage-specific chorion genes are discussed in light of these findings and the known effects of Gr^B on stage-specific protein synthesis.     

Evolution of a multigene family of chorion proteins in silkmoths.

The evolution of the A family of chorion genes was examined by comparing new protein and DNA sequences from the silkmoths Antheraea pernyi and Bombyx mori with previously known sequences from Antheraea polyphemus. The comparisons indicated that the A family and its major subfamilies are ancient and revealed how parts of the genes corresponding to distinct regions of the protein structure have evolved, both by base substitutions and by segmental reduplications and deletions.     

Evolution of two major chorion multigene families as inferred from cloned cDNA and protein sequences

Complete or partial sequences are reported from six chorion cDNA clones of the silkmoth Antheraea polyphemus. The proteins encoded belong to the two major chorion protein classes, A and B, each of which is encoded by a multigene family. The sequence comparisons define some major features of the families and suggest how these genes may be evolving. Deletions and insertions might be involved in expanding or contracting internally repetitive regions. Sequence divergence is localized, thus defining sequence domains of distinct evolutionary properties and presumably distinct functions.     

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.     

Molecular evolution of the cecropin multigene family in silkworm Bombyx mori

Cecropins constitute one of the largest and most potent immune protein families found in insect species with diversified numbers and features. In view of the large number of cecropin proteins existing with much sequence variations among them, an overview of the multigene cecropin family in silkworm Bombyx mori was attempted in this study. Cecropin encodes an inducible 64 residue anti-bacterial peptide and was clustered into two groups; first group viz. A and second group including B, D, E and Enbocin. Cecropin A consisted of two sub-groups located on chromosome number 6 of B.mori genome. Cecropin B consisted of six sub-groups, cecropin D and E of one each and Enbocin of two. The second sub-group formed in tandem array of multigene family locus over a length of 78.62 kb on chromosome number 26 in B.mori genome and was organized in positive as well as opposite orientation. The results indicated that cecropin B genes were organized in a close cluster with the intergenic sequence ranging from 1366 bp to 23526 bp. Interestingly a distantly related cecropin E was also located within the cecropin B multigene locus. Similarly distant members like cecropin D and Enbocin were also located in the 3' region of cecropin B locus. The maximum intergenic region of 23526 bp observed between Cecropin D and Enbocin indicates that the two genes were distantly evolved. The phylogenetic analysis clearly indicates a positive correlation between the clusters and physical location on the chromosome, as the length of the intergenic region plays a major role to create newer cecropin families. EST database analysis suggests that most of the cecropin A members were expressed in the microbial fat body while, the cecropin B was equally expressed in fat body and other target tissues. The signal peptides were conserved in all the twelve paralogous gene sequences.     

Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families

A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stage-specific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.     

Sequence Identity in an Early Chorion Multigene Family Is the Result of Localized Gene Conversion

The multigene families that encode the chorion (eggshell) of the silk moth, Bombyx mori, are closely linked on one chromosome. We report here the isolation and characterization of two segments, totaling 102 kb of genomic DNA, containing the genes expressed during the early period of choriogenesis. Most of these early genes can be divided into two multigene families, ErA and ErB, organized into five divergently transcribed ErA/ErB gene pairs. Nucleotide sequence identity in the major coding regions of the ErA genes was 96%, while nucleotide sequence identity for the ErB major coding regions was only 63%. Selection pressure on the encoded proteins cannot explain this difference in the level of sequence conservation between the ErA and ErB gene families, since when only fourfold redundant codon positions are considered, the divergence within the ErA genes is 8%, while the divergence within the ErB genes (corrected for multiple substitutions at the same site) is 110%. The high sequence identity of the ErA major exons can be explained by sequence exchange events similar to gene conversion localized to the major exon of the ErA genes. These gene conversions are correlated with the presence of clustered copies of the nucleotide sequence GGXGGX, encoding paired glycine residues. This sequence has previously been correlated with gradients of gene conversion that extend throughout the coding and noncoding regions of the High-cysteine (Hc) chorion genes of B. mori. We suggest that the difference in the extent of the conversion tracts in these gene families reflects a tendency for these recombination events to become localized over time to the protein encoding regions of the major exons.     

The silkmoth late chorion locus. I. Variation within two paired multigene families

The 140 X 10(3) base late chorion locus of Bombyx mori contains two 15-member multigene families arranged in tightly linked pairs, which are divergently transcribed (the high-cysteine A (HcA) and the high-cysteine B (HcB) families). Previous DNA hybridization experiments have indicated that all members of these gene families contain a complex pattern of shared sequence variation. The sequence analysis in this paper involving all 15 gene pairs allows a comprehensive examination of the nature of this variation. Average sequence homology between gene pairs is: 95% for the protein-encoding regions; 93% for the common 272 base-pair 5' flanking region; 87% for the introns; and 88% for the 3' untranslated regions. Considering the great degree of sequence homology in the coding regions, an unexpectedly high level of variation is found in the deduced protein sequences. Over 50% of the nucleotide substitutions in the protein-encoding regions lead to amino acid replacements, most of which involve a change in charge or effect the secondary structure of the protein. In addition, significant differences in length between the proteins occur in the carboxyl-terminal arm. In both families, the major portion of this arm is composed of Cys-Gly-Gly and Cys-Gly subrepeats forming a (Cys-Gly-Gly)2-(Cys-Gly)2 major repeat. Differences in the number of complete and partial repeats results in deduced protein sequences that contain arms varying from 32 to 54 amino acid residues for members of the HcA family and 14 to 88 residues for the HcB family. The high level of variation in protein composition indicates a lack of strong selective pressure. We suggest the high level of DNA sequence homology maintained by these genes in the coding as well as in the non-coding regions is the result of sequence exchange between family members.     

Gene evolution and regulation in the chorion complex of Bombyx mori. Hybridization and sequence analysis of multiple developmentally middle A/B chorion gene pairs

Twenty-two pairs of chorion genes belonging to the A and B multigene families have been characterized and mapped within two segments of a 320 kb (1 kb = 10(3) bases or base-pairs) chromosomal walk in the domesticated silkmoth Bombyx mori. Eighteen of the gene pairs belong to two groups that are typified by the previously characterized A/B.L12 and A/B.L11 chorion gene pairs, and are defined by two respective types of short (approx. 280 base-pairs) bidirectional promoter sequences. In the chromosome, the L12-like and L11-like pairs are interspersed with each other and with the remaining four gene pairs, which have unrelated promoter sequences. We have sequenced the promoter regions and adjacent small exons of all L12-like and L11-like A and B genes in the walk. The L12-like promoters are highly conserved, whereas L11-like promoters are somewhat more variable. Reconsideration of previous data on RNA accumulation and disappearance during choriogenesis, in the light of the sequences, indicates that L12-like genes are developmentally early-middle, while L11-like genes correspond to two developmental subgroups, middle I and middle II. Sequence comparisons of all these promoters, as well as the previously characterized promoters of the developmentally late HcA and HcB genes, identify short elements of possible regulatory significance. The sequences, as well as extensive cross-hybridization analysis with short probes derived from the reference A/B.L12 gene pair, under carefully controlled conditions of stringency, indicate the occurrence of sequence transfers among A or B genes. These sequence transfers, which could result from gene conversions or unequal crossovers, are less abundant than in the HcA and HcB families, but do result in a patchwork of similarities and differences in the A and B genes. The transfers appear to be least frequent between the moderately divergent A genes that belong to different temporal classes, while the L12-like and L11-like B genes appear to be extensively homogenized in sequence.     

A walk in the chorion locus of bombyx mori

We have recovered overlapping clones that represent in the aggregate a contiguous segment of chromosomal DNA 270 kb in length, or probably one third of the chorion locus of Bombyx mori. Approximately 70 genes have been identified, the majority of which are arranged in coordinately expressed pairs. The nonidentical genes expressed in the late period of choriogenesis are clustered within a single, 130 kb region, which is flanked by regions containing genes that are active during the middle developmental period. The late genes encode two families of high-cysteine proteins; the evolutionarily persistent clustering of these families contrasts sharply with the extensive sequence diversification of the structural genes and their flanking DNA elements. We discuss the possible regulatory significance of the clustered arrangement, as well as certain features of multigene family evolution.     

GrB deletion of the chorion locus of the silkmoth Bombyx mori. Localization of the left breakpoint and isolation of the deletion junction

In the silkmoth Bombyx mori, choriogenesis occurs through the developmentally controlled deposition of several related classes of chorion proteins onto the oocyte by surrounding follicular cells. In the GrB mutant strain, a distinctive family of proteins (Hc) normally expressed late in choriogenesis, as well as several proteins of middle development specificity, are missing due to the deletion of the corresponding genes from the chorion locus. In addition, a smaller set of proteins normally confined to mid-choriogenesis is found to be prolonged in expression in homozygote mutant but not heterozygote individuals. To elucidate the molecular organization of the chorion locus in the GrB genotype, we scanned a part of the wild-type locus represented by a chromosomal walk of 270,000 bases through library screening and genomic DNA hybridizations using a series of unique probes. A chromosomal clone, GrB4, whose sequences showed the expected characteristics of the deletion junction, was isolated from a partial EcoRI library of mutant genomic DNA. Through comparative hybridizations, mapping and sequencing, the precise location of one of the deletion breakpoints was identified on one of the clones mapping in the characterized part of the wild-type locus. Attempts to locate the other breakpoint in wild-type DNA and to extend the structural characterization past the deletion junction through chromosomal walking were unsuccessful, due to the apparent absence of these sequences from libraries of wild-type and mutant genomic DNA, respectively. Hybridizations of the deletion region on clone GrB4 to cDNA derived from follicular RNA indicate that no gene sequences are directly interrupted by the deletion, and reveal the presence of a gene sequence of unknown function 1000 to 5000 bases to the right of deletion junction.     

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.     

The chitinase gene of the silkworm, Bombyx mori, contains a novel Tc-like transposable element

We have determined the cDNA sequence and the genomic organization of the chitinase gene of the silkworm, Bombyx mori. The cDNA encodes 544 amino acids having 83% amino acid homology to the chitinase of the tobacoo hornworm, Manduca sexta. The total length of the gene is larger than 25 kilobase pairs, and it is separated into 11 exons. The intron-exon boundaries are all in accordance with the GT-AG rule. Also, the TATA box sequence was found in the 5' upstream region of the gene, and the gene is mapped on the seventh chromosome. A novel DNA type transposon that shows similarity to the Tc-like element was found in the third intron in some strains of B. mori; other strains, however, lack this element in the same intron. This element has long terminal inverted repeats, presumably encodes a transposase of about 340 amino acids with a DDE motif, and has an amino-terminal domain with a strong nuclear localization function. Seven other transposable elements with homologous but distinct sequences were isolated from the B. mori genome. Together with plaque hybridization results, our findings suggest that these novel elements exist in multiple copies constituting a new Tc-like transposable element family in the silkworm genome.     

Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA

Molecular phylogeny of some of the economically important silkmoths was derived using three mitochondrial genes, 12S rRNA, 16S rRNA, and COI, and the control region (CR). Maximum likelihood (ML) analyses showed two distinct clades, one consisting of moths from Bombycidae family and the other from Saturniidae family. The mitochondrial CR showed length polymorphisms with indels. The ML analyses for complete mitochondrial genome sequences of Bombyx mori (strains Aojuku, C108, Backokjam, and Xiafang), Japanese and Chinese strains of B. mandarina (Japanese mandarina and Chinese mandarina) and, Antheraea pernyi revealed two distinct clades, one comprising of B. mori strains and the other with B. mandarina, and A. pernyi forming an outgroup. Pairwise distances revealed that all of the strains of B. mori studied are closer to Chinese than to Japanese mandarina. Phylogenetic analyses based on whole mitochondrial genome sequences, the finding of a tandem triplication of a 126bp repeat element only in Japanese mandarina, and chromosome number variation in B. mandarina suggest that B. mori must have shared its recent common ancestor with Chinese mandarina. Another wild species of the Bombycidae family, Theophila religiosa, whose phylogenetic status was not clear, clustered together with the other bombycid moths in the study. Analysis of the interspecific hybrid, A. proylei gave evidence for paternal inheritance of mitochondrial DNA.