Nonuniform evolution of duplicated,developmentally controlled chorion genes in a silkmoth

Summary We report the sequence of A/B.L1, a tightly linked pair of genes from the A and B chorion families inBombyx mori. Comparison with the previously characterized A/B.L11 and A/B.L12 pairs from the same species reveals moderate sequence divergence, which is clearly nonuniform. Although the average divergence of A/B.L12 from the other two pairs is more than double that between A/B.L11 and A/B.L1, the ratio differs by more than 30-fold in different DNA regions. One domain of the A gene is highly divergent between A/B.L12 and A/B.L1 or A/B.L11, but essentially invariable in the latter two. In well-aligned DNA segments, nearly all mutated sites (111/112) show variants shared by two of the three sequences, in 42% of the cases between the more distantly related pairs (A/B.L12 and either A/B.L1 or A/B.L11). Eight of the variants shared by distantly related pairs are clustered within 51 bp, suggesting the possibility that they arose through sequence transfers between gene pairs, rather than being primitive or resulting from independent mutations. The short intergenic, putatively regulatory DNa of the developmentally middle A/B.L1 and A/B.L11 pairs resembles that of the late HcA/HcB pairs, particularly in patches that may correspond tocis-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.     

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 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.     

Silk moth chorion pseudogenes: Hallmarks of genomic evolution by sequence duplication and gene conversion

The part of the genetic locus of the domesticated silk moth,Bombyx mori, in which high cysteine (Hc) chorion genes of late developmental specificity reside contains regions encompassing genelike sequences which exhibit properties distinct from those of functional Hc genes. One of these regions has been characterized and shown to contain a chorion pseudogene, ψHcB.15, which shares pronounced similarities with a transcribed chorion pseudogene, ψHcB.12/13, which was characterized previously. Both pseudogenes are homologous to HcB chorion genes but bear multiple single nucleotide substitutions and short segmental mutations (insertions and deletions) which introduce translational frame shifts and termination codons in the coding regions. Structural characteristics unique to the two pseudogenes suggest that ψHcB.15 was generated first from a functional HcB gene and gave rise subsequently to ψHcB 12/13 as a result of a sequence duplication event. The two pseudogenes can be distinguished from each other by the presence of distinct regions of similarity to the consensus sequence of functional HcB genes which appear to have arisen from gene-conversionmediated correctional events. These findings lend support to the hypothesis that chorion pseudogene sequences represent reservoirs of genetic information that participates in the evolution of the chorion locus rather than relics of inactivated genes passively awaiting extinction. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Loss of phylogenetic information in chorion gene families of Bombyx mori gene conversion

The silkmoth chorion has provided a stimulating model for the study of evolution and developmental regulation of gene families. Previous attempts at inferring relationships among chorion sequences have been based on pairwise comparisons of overall similarity, a potentially problematic approach. To remedy this, we identified the alignable regions of low sequence variability and then analyzed this restricted database by parsimony and neighbor-joining methods. At the deepest level, the chorion sequence tree is split into two branches, called "alpha" and "beta." Within each branch, early- and late-expressing genes each constitute monophyletic groups, while the situation with middle-expressing genes remains uncertain. The HcB gene family appears to be the most basal beta-branch group, but this conclusion is qualified because the effect of gene conversion on branching order is unknown. Previous studies by Eickbush and colleagues have strongly suggested that ErA, HcA, and HcB families undergo gene conversion within a gene family, whereas the ErB family does not. The occurrence of conversion correlates with a particular tree structure; namely, branch lengths are much greater at the base of the family than at higher internodes and terminal branches. These observations raise the possibility that chorion gene families are defined by gene conversion events (reticulate evolution) rather than by descent with modification (synapomorphy).      

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori

Summary Three -type early chorion gene copies (6F76.1, 6F76.2, and 6F76.3) are dispersed in the late region of chorion locus Chl-2. Detailed analysis of the 5-flanking region and the intron of 6176.1 shows that they contain sequences that are homologous to Bombyx mori Bm l repeat elements. Interestingly, the Bm l -type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6176.1 5-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion -gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.Offprint requests to: G. Rodakis     

The human H2A and H2B histone gene complement

Sequences and expression patterns of newly isolated human histone H2A and H2B genes and the respective proteins were compared with previously isolated human H2A and H2B genes and proteins. Altogether, 15 human H2A genes and 17 human H2B genes have been identified. 14 of these are organized as H2A/H2B gene pairs, while one H2A gene and three H2B genes are solitary genes. Two H2A genes and two H2B genes turned outto be pseudogenes. The 13 H2A genes code for at least 6 different amino acid sequences, and the 15 H2B genes encode 11 different H2B isoforms. Each H2A/H2B gene pair is controlled by a divergent promoter spanning 300 to 330 nucleotides between the coding regions of the two genes. The highly conserved divergent H2A/H2B promoters can be classified in two groups based on the patterns of consensus sequence elements. Group I promoters contain a TATA box for each gene, two Oct-1 factor binding sites, and three CCAAT boxes. Group II promoters contain the same elements as group I promoters and an additional CCAAT box, a binding motif for E2F and adjacent a highly conserved octanucleotide (CACAGCTT) that has not been described so far. Five of the 6 gene pairs and 4 solitary genes with group I promoters are localized in the large histone gene cluster at 6p21.3-6p22, and one gene pair is located at 1q21. All group II promoter associated genes are contained within the histone gene subcluster at D6S105, which is located at a distance of about 2 Mb from the major subcluster at 6p21.3-6p22 containing histone genes with group I promoters. Almost all group II H2A genes encode identical amino acid sequences, whereas group I H2A gene products vary at several positions. Using human cell lines, we have analyzed the expression patterns of functional human H2A/H2B gene pairs organized within the two histone gene clusters on the short arm of chromosome 6. The genes show varying expression patterns in different tumor cell lines.     

Structural features of B family chorion sequences in the silkmoth Bombyx mori, and their evolutionary implications.

Partial protein sequences, and DNA sequences of corresponding cDNA and genomic clones were obtained and analyzed to reveal the primary structural features of major, developmentally middle or late components of the B chorion multigene family in Bombyx mori. Comparisons with other types of sequences confirm and clarify the tripartite domain structure of chorion proteins. Glycine-, leucine- and tyrosine-containing, tandemly repetitive peptides form the bulk of the amino-terminal and carboxy-terminal domains ('arms'). Extensive sequence homologies suggest a common evolutionary origin for the amino-terminal arms of some B. mori B sequences and the corresponding portions of members of a different (A) chorion multigene family in Antheraea polyphemus, a distantly related silkmoth.