Organization and developmental timing of the Bombyx mori chorion gene clusters in strain C108

We have standardized the map of chorion structural gene clusters in Bombyx mori strain C108 by analyzing quantitative and qualitative chorion electrophoretic markers in recombinant progeny from four independent crosses. In all we assigned 22 markers to three gene clusters, representing about one-third of the total number of chorion genes: 2 to Ch 1, 9 to Ch 2, and 8 to Ch 3. Three additional markers belong either to Ch 7 or Ch 2. By referring to published chorion protein synthesis patterns, we show that the clusters are restricted in their developmental specificities: Ch 3 appears to be an early locus, carrying all of the mapped early markers (4) and half of the early middles (3/6), while Ch 1 and Ch 2 carry predominantly middle (4/5) and all late, Hc (6) markers, along with some early middle markers (3). We cite evidence to show that Ch 1 and Ch 2 compose the left and right halves of a single gene cluster, which we formally designate as Ch 1–2.     

RFLP analysis of chromosomal fragments in genetic mosaic strains of Bombyx mori

Mottled striped (p^Sm), a genetic mosaic strain for larval body marking of the silkworm, Bombyx mori, contains a small chromosomal fragment generated by breakage near the end of the 2nd chromosome. This fragment carries the striped marking (p ^S ) gene and part of the chorion gene clusters. To determine the structural features of this fragmented chromosome, we studied the organization of the chorion genes on the fragment using restriction fragment length polymorphism (RFLP) analysis. Tow of three classes of chorion gene family probes detected RFLPs in the two phenotypes, p^Sm (p/p/p ^S ), and p (p/p), which had lost the fragment, segregated among the siblings of a mottled striped strain. Although hybridization patterns were basically identical between them, one or two additional bands were always observed associating with the mosaic phenotype (p^Sm). This suggests that the additional bands correspond to extra copies of chorion genes on the chromosomal fragment, which have a different structure from ones on the intrinsic 2nd chromosomes. Such heterogeneity of chorion genes may have been maintained since the beginning of mosaic induction, due to the absence of recombinational events between the two chromosomes. We are unable to detect any RFLPs by hybridization with the early class of chorion genes, implying that chromosomal breakage might have occurred between the two chorion clusters, Chl-2 and Ch3, which are located approximately 4 cM apart from each other. Based on RFLP analyses for two independent mosaic strains (788 and 872), we postulate a common chromosomal origin with independent breakpoints and construct structural models for the two kinds of chromosomal fragments.     

Organization of the Chorion Genes of BOMBYX MORI, a Multigene Family. I. Evidence for Linkage to Chromosome 2

The chorion genes of silkmoths comprise a multigene family that codes for 50 or more highly specialized structural proteins found in the eggshell. A detailed study of the chromosomal organization of these genes was initiated, using inbred stocks of Bombyx mori as a source of electrophoretic variants for genetic markers. Chorion protein patterns were screened on thin-slab polyacrylamide isoelectric focusing gels. A wide range of polymorphism was observed between stocks. However, isoelectric focusing patterns obtained within a stock were nearly homogeneous, indicating that inbreeding has produced a high degree of homozygosis. Testcrosses were carried out to examine the linkage relationships between electrophoretic markers in four inbred stocks. One race (C108) was selected as a standard against which to compare the inheritance of the variants found in the other three stocks. Chorion markers behaved like codominant Mendelian traits in F₁ crosses. A total of 15 out of 16 C108 markers cosegregated in subsequent testcrosses, indicating that they are linked. These genes were mapped to the second chromosome, using markers Gr and Y.     

Evolution of chorion structural genes and regulatory mechanisms in two wild silkmoths: A preliminary analysis

Summary We report a preliminary analysis of structural and regulatory evolution of the A and B chorion gene families in two wild silkmoths,Antheraea pernyi andAntheraea polyphemus. Homospecific and heterospecific dot hybridizations were performed between previously characterizedA. polyphemus complementary DNA clones and total or stage-specific follicular mRNAs from the two species. The hybridization patterns indicated substantial interspecies changes in the abundance of corresponding mRNA sequences (heteroposic evolution) without substantial changes in their developmental specificities (heterochronic evolution). In addition, the proteins encoded in the two species by corresponding mRNAs were determined by hybrid-selected translation followed by electrophoretic analysis. The results suggested that the proteins evolve in size, presumably through internal deletions and duplications.     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

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     

Genetic variability of leaf esterases in Triticum aestivum L. 2n=6x=42

Summary Starch gel electrophoresis with two different buffer systems and several substrates and inhibitors have been used to study the electrophoretic variability of esterases in leaves of cultivars of Triticum aestivum. Each one of the buffer systems showed different levels of variability, according to the electrophoretic patterns. At the same time green and etiolated leaves showed different patterns in each buffer system. The variability was dependent upon the developmental stage of the leaves. According to the results from chromosomal location, the genes controlling esterases in green leaves were located in homoeology group 3, while the genes controlling esterases in etiolated leaves were in homoeology group 6. But both esterase isozymes showed a similar electrophoretic migration and a similar reponse to substrates and inhibitors. The possible origin of both sets of genes due to an interchromosomal duplication is discussed.     

Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes)

One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.     

DNA sequence of a 3.8 kilobase pair region controlling Drosophila chorion gene amplification

During Drosophila oogenesis, two clusters of chorion genes and their flanking DNA sequences undergo amplification in the ovarian follicle cells. Amplification results from repeated rounds of initiation and bidirectional replication within the chorion gene regions, possibly from a single origin, producing nested replication forks. Previously we have shown that following reintroduction into the Drosophila genome, a specific 3.8 kilobase pair DNA segment from the amplified third chromosome domain could induce developmentally regulated amplification at its site of insertion. Here we present the complete nucleotide sequence of this amplification control element and of genes encoding the chorion structural proteins s18-1 and s15-1, which are contained within it. Sequences that may be involved in the regulation of chorion gene amplification and expression are identified.     

Drosophila bearing the ocelliless mutation underproduce two major chorion proteins both of which map near this gene.

Two bands of putative Drosophila chorion mRNA, E3 and E4, have been shown to hybridize in situ near 7E11 (Spradling and Mahowald, 1979) within a region known to contain a gene, ocelliless, which may be involved in chorion production (Johnson and King, 1974). We have investigated the synthesis of “chorion mRNAs” and chorion proteins in flies carrying this mutation. A reduction of labeling of both E3 and E4 was observed in stage 12 egg chambers from homozygous ocelliless females. In addition, they produce mature oocytes which contain greatly reduced amounts of several major chorion proteins, including those normally produced in stage 12, c36 and c38. To investigate whether the reduction was due to a direct effect of the mutation, the genes for these proteins were mapped. Recombination analysis using electrophoretic variants of c36 and c38 showed that both proteins are coded on the X chromosome at a site between crossveinless and vermillion. Further mapping with the deficiency chromosomes Df(1)KA14 and Df(1)RA2 narrowed the region containing the structural genes to a 16 band region between 7E10 and 8A4. The ocelliless gene, as well as the site of in situ hybridization, are located within this same interval. In normal ovarian follicles, both c36 and c38 are produced in equal amounts and with the same developmental specificity (Waring and Mahowald, 1979). In the mutant, both are reduced to a similar extent. In oc⁺/oc heterozygotes, both the c36 and c38 genes on the mutant chromosome produce much less product than the corresponding genes on the oc⁺ chromosome. The cis-acting nature of the ocelliless mutation suggests that it may disrupt sequences involved in controlling the expression of the structural information for these proteins.     

Tissue specific isozyme expression in three species of icterinae (Aves: Emberizidae)

Tissue specific banding patterns were examined for ten enzyme systems in three species of the subfamily Icterinae, family Emberizidae — Quiscalus quiscula, Agelaius phoeniceus and Molothrus ater. Ten tissues were compared. Generally, tissue specific patterns observed were remarkedly similar as compared to other taxa, across the three species over all enzyme systems examined, indicating low levels of divergence in regulatory genes. This concurs with other observations of conservative avian protein evolution as indicated by small comparative electrophoretic distances between species.     

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster

Summary Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1,3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controlls cell sensitivity to ecdysterone is also necessary for female fertility. Data on the locations of lesions affecting female fertility indicate that at least two elements at the ecs locus are essential for this function: a cis-acting distal zone with no effect on viability and a sequence within the essential part of the ecs locus. A defect in either of these zones or their separation by chromosomal rearrangement leads to female sterility.     

Coding and potential regulatory sequences of a cluster of chorion genes in Drosophila melanogaster

We have characterized at the nucleotide level a 4.8-kilobase pair segment of the third chromosome of Droophila melanogaster, which contains a cluster of three chorion genes, s 18-1, s 15-1 and s 19-1. These genes are tandemly oriented and share the same basic organization: a small and a large exon separated by a short intron in the signal peptide region. In the coding region, limited similarities at the DNA and protein level suggest a common but distant evolutionary origin. The flanking sequences were searched for elements that might be involved in controlling the tissue-specific and temporally regulated expression and the selective amplification of the chorion genes. A good candidate for a cis-regulatory element is the hexamer, TCACGT, which is found in all three genes in a highly significant position, 23 to 27 nucleotides upstream of the TATA-box, accompanied by additional, less exact similarities. Palindromes and short inverted repeats that are found in the vicinity of their complement are non-uniformly distributed: they are most concentrated in the 3 flanking part of all three genes, in and near regions of unusually high A and T content. The highest number of dyad symmetries, remiiscent of sequences that function as viral replication origins, is found associated with the T- and A-rich regions between genes s18-1 and s15-1.     

PHOSPHOGLUCOMUTASE AND ISOCITRATE DEHYDROGENASE GENE DUPLICATIONS IN LAYIA (COMPOSITAE)

Genetic analysis of isozyme segregation patterns in Layia (Compositae) showed that cytosolic phosphoglucomutase isozymes are encoded by duplicated genes, and that the cytosolic NADP-dependent isocitrate dehydrogenase isozymes are encoded by duplicated genes in species with haploid chromosome numbers of n = 7 and triplicated genes in those with n = 8. The duplicated genes specifying both isozymes assorted independently in all species tested. An electrophoretic survey of phosphoglucomutase in diploid species representing six additional genera of Madiinae, the subtribe to which Layia is assigned, revealed that Achyrachaena, Calycadenia, Hemizonia, Holocarpha, and Madia all possessed duplicated genes. In Lagophylla, one species also had duplicated genes for the isozyme but a second species did not, a loss probably resulting from mutation or chromosomal deletion. The phosphoglucomutase duplication characterizes nearly the entire subtribe and may prove useful to identify phylogenetic relationships between the Madiinae and other subtribes.     

Heterospecific combinations of germ cells and gonadal soma betweenDrosophila melanogaster,D. mauritiana andD. ananassae

Summary We transplanted pole cells betweenDrosophila melanogaster, D. mauritiana andD. ananassae to investigate the ability of germ cells to develop in the gonad of a heterospecific host, and to study the interaction between somatic follicle cells and the cells of the germ line in producing the species-specific chorion. FemaleD. mauritiana germ cells in aD. melanogaster ovary produced functional eggs with normal development potential. The same is true for the reciprocal combination. FemaleD. ananassae pole cells in aD. melanogaster host only developed to a very early stage and degenerated afterwards. None of the interspecific combinations of male pole cells led to functional sperm. We could not determine at what stage the transplanted male pole cells were arrested. The cooperation of follicle cells and the oocyte-nurse cell complex in producing the chorion was studied using the germ-line-dependent mutationfs(1) K10 ofD. melanogaster, which causes fused respiratory appendages and an abnormal chorion morphology. Wild-type femaleD. mauritiana germ cells in a mutantfs(1) K10 D. melanogaster ovary led to the production of wild-type eggs withD. melanogaster-specific, short respiratory appendages. On the other hand,D. melanogaster fs(1) K10 germ cells in aD. mauritiana ovary induced the formation of eggs with mutant fused appendages which were, however, typicallyD. mauritiana in length. When.D. mauritiana pole cells developed in aD. melanogaster ovary, the chorion exhibited a new imprint pattern that differs from both species-specific patterns.     

Comparison of Coding and Spacer Region Sequences of Chromosomal rRNA-Coding Genes of Two Sequevars of Pneumocystis carinii

Two distinct sequevars, denoted Pc1 and Pc2, of the opportunistic pathogen Pneumocystis carinii have been previously identified based on the sequence of their 26S rRNA genes, the location of group I self-splicing introns and pulsed field electrophoretic patterns of chromosomal DNA. This study shows that the sequences of 16S and 5.8S rRNA genes also vary between these sequevars, and that greater variation was seen in the internal transcribed spacer regions. Polymerase chain reaction and restriction analysis can distinguish between these sequevars.     

Patterns of protein synthesis in oocytes and early embryos of Rana esculenta complex

Summary We have used isotopic labelling and both one-and two-dimensional electrophoretic procedures to analyse the protien synthesis patterns in oocytes and early embryos of three phenotypes of the European green frogs. The results demonstrated that protein patterns of Rana ridibunda and R. esculenta are identical, but that they differ from those of R. lessonae. Progeny of the lethal cross R. esculenta × R. esculenta showed a distinct delay in the appearance of stage-specific proteins during early embryogenesis. The heat-shock response of R. ridibunda and R. esculenta oocytes was found to be identical, but different from that of Xenopus laevis. The implications of these findings, with respect to hybridogenesis in R. esculenta complex and variations in the regulations of heat shock genes in different amphibian species, are discussed.     

Taxonomic identification of Drosophila nasuta subgroup strains by glue protein analysis

Protein fractions of salivary glands were analyzed from 30 wildtype strains of eight species belonging to the Drosophila nasuta subgroup by SDS-polyacrylamide gel electrophoresis. The electrophoretic patterns indicated several prominent bands which could be shown to represent the major glue protein fractions. The glue protein fractions are species-specific as well as wildtype strain-specific. Wildtype strain specificities are characterized by variations of the species-specific patterns. The patterns of the different wildtypes, species, and hybrids were used for taxonomic identification within the nasuta subgroup, in which the females are morphologically indistinguishable and the males differ only by the markings of their frons. The hybrids provide evidence for gonosomal as well as autosomal linkage of individual genes coding for the major glue protein fractions.     

Chorion genecis-regulatory DNA restricts tissue specificity of reporter gene expression in transformedDrosophila

P element mediated germ-line transformation was used to study the developmental specificity ofDrosophila chorion gene regulatory sequences directing expression of the bacterial reporter genes for chloramphenicol acetyltransferase (CAT) and -galactosidase (lacZ). DNA fragments containing 5 flanking plus the entire 5 untranslated and the beginning of the coding region of either thes36 or thes15 chorion gene are able to confer on the reporter genes normal tissue as well as temporal specificity of expression, exclusively in the ovary of transformed female flies. However, if 5 untranslated and coding regions are omitted, normal ovarian expression is maintained but tissue specificity is relaxed: expression of the reporter gene is detected both in the ovary and in specific non-ovarian tissues of transformed females and males. The evidence suggests that the missing 5 untranslated and coding sequences may include negative elements that normally suppress expression in non-ovarian tissues, and that these putative elements are distinct from those that prevent premature expression in the ovarian follicles. The exact location of ectopiclacZ expression within the internal male genitalia depends on the constellation of 5 flanking chorion regulatory sequences included in theP element constructs. Ectopic expression of theCAT gene in the male genitalia unders15 promoter control can be abolished by mutating the hexamer TCACGT, a sequence previously shown to be essential for the normal expression of this chorion gene in the ovary.by A. Spradling