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.     

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     

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     

Sequence analysis of a small early chorion gene subfamily interspersed within the late gene locus in Bombyx mori

A comprehensive sequence analysis of three early chorion genes (6F6.1, 6F6.2, 6F6.3) which form a small subfamily is presented. Two main features characterize this subfamily: (1) the 6F6 gene copies are -branch genes and, unlike typical chorion genes which are organized in divergent gene pairs, they are unpaired, and (2) they are not clustered in genetic locus Ch3 but are dispersed in Ch1-2, which is about 3 to 4 centiMorgans away and contains middle and late chorion genes. Sequence comparisons show that members of this subfamily exhibit high identity values in their major coding region (94–96%) and that similarities also extend, but to a lesser degree, into their noncoding regions. The putative 6F6 promoter regions have no significant similarities with the corresponding regions of other early -genes but quite surprisingly share common elements with middle and late genes. The main difference among the 6F6 gene introns is the presence of inserted sequences: the insert into 6F6.2 (IR; 248 bp) is flanked by a 102–103-bp inverted repeat, while those into 6F6.1 (FIB; 184 bp) and 6F6.3 (HOPE; 951 bp) are carried by a partial Bm1 element. HOPE has features of a non-LTR retrotransposable element. Preliminary experiments indicate that the copy number of IR and HOPE in the Bombyx mori genome is about 5,000 and 20,000, respectively. The great similarity of 6F6 genes cannot be accounted for by selective pressure but rather appears to be the result of gene-conversion-like events, which are supposed to operate frequently in middle and late chorion genes but not in other known early -genes. Using the relative position and orientation of the 6F6 gene copies, it is possible to propose an evolutionary scheme for the formation of chorion locus Ch1-2. Correspondence to: G.C. Rodakis     

Sequence variation, differential expression and chromosomal location of rice chitinase genes

Rice chitinases are encoded by a small multigene family. To clarify the overall organization of rice chitinase genes, we have isolated and characterized the genes Cht-1, Cht-2 and Cht-3. Although all the three genes encode class I chitinase, the nucleotide sequences of the coding regions of Cht-1 and Cht-3 are very similar (90%), while that of Cht-2 is clearly more divergent (78%). Only Cht-2 has a 130 by intron and encodes a C-terminal peptide sequence similar to that known to function as a vacuolar targeting signal. In 5 flanking regions of Cht-1 and Cht-3, but not of Cht-2, conserved sequences (GGCCGGCYGCCCYAG) were found. Related sequences were found also in the 5 flanking regions of another chitinase gene and a -glucanase gene which has also been reported to be stress-induced in rice. RNA blot hybridization analysis demonstrated that the stress-induced expression patterns of the Cht-1 and Cht-3 genes are similar, but quite different from that of Cht-2. However, all three genes are active in unstressed roots. By restriction fragment length polymorphism (RFLP) linkage analysis, Cht-1 and Cht-3 were mapped onto chromosome 6 and shown to be closely linked (0.8 cM). Cht-2 was mapped onto chromosome 5. All these features suggest that the expression patterns of rice class I chitinase genes may be correlated with their levels of sequence divergence and their chromosomal location.     

Structure and expression of kin2, one of two cold- and ABA-induced genes of Arabidopsis thaliana

We report the isolation of the second member, kin2, of a family of two cold-inducible genes of Arabidopsis thaliana. The proteins corresponding to the two genes have similarities to the small antifreeze proteins from Winter flounder. Kin1 and kin2 are organized in a close tandem array in the genome of a. thaliana. Both have three exons separated by introns with approximately the same length and location. The coding regions are highly conserved while the introns and especially the 3 flanking sequences of the mRNAs have diverged. The kin1 and kin2 genes are coordinately regulated in the cold. Unlike kin1, the kin2 mRNA has a detectable basal level, and accumulates to a higher level during acclimation. Both mRNAs are induced by 10 M ABA but only kin2 responds strongly to drought and salinity stresses.     

Molecular characterization of two clusters of genes encoding the Type I CAB polypeptides of PSII in Nicotiana plumbaginifolia

A Nicotiana plumbaginifolia genomic library in the phage Charon 34 was used to isolate and characterize 7 full-length genes and part of an 8th gene encoding chlorophyll a/b-binding (CAB) polypeptides. These genes are arranged in two clusters. All the genes within the clusters are arranged in opposite orientation to their neighbours. The nucleotide sequences of two genes, one from each cluster, show that both genes, designated Cab-E and Cab-C, encode very similar proteins (95.9% of homology) corresponding to type I photosystem II polypeptides. Southern blot analysis suggests that at least 19 CAB genes encoding type I PSII CAB polypeptides are present in the N. plumbaginifolia genome. We also describe the presence within the N. plumbaginifolia genome of CAB genes encoding PSII type II CAB polypeptides and PSI type I CAB polypeptides. The sequences of the 5 flanking region of three different CAB genes (Cab-E, Cab-C, and CAB-F) were determined. Two of them (Cab-C and Cab-F) share extensive homology, whereas the Cab-E promoter shows homology to Cab-C and Cab-F only in a unique region extending from the CAAT box to the TATA box. This conserved sequence is also found in the same position in promoters of CAB genes encoding type I PSII polypeptides from other plant species.Abbreviations CAB chlorophyll a/b-binding protein - bp base pair(s) - kb 1 000 bp     

Evolution of chorion gene families in Lepidoptera: Characterization of 15 cDNAs from the gypsy moth

Fifteen unique chorion protein-encoding cDNAs from gypsy moth have been completely sequenced. These sequences are encoded by a family of genes, based on pairwise similarity values of 78–100% within a 225-nt region. Pairwise comparisons and maximum parsimony analysis strongly support the existence of two clusters of 11 and four sequences each, called noel and noc2. While noc2 consists of two subclusters, there is little character support for subclusters within noel. The highly localized character-state distribution on the parsimony tree in gypsy moth is reminiscent of that in Bombyx mori, specifically for those chorion families that have been shown to undergo gene conversion. Gene conversion thus becomes a reasonable explanation for the homogeneity of noel sequences and for their distinctness from noc2. The relationship between the two major clusters of chorion sequences in gypsy moth (noel, noc2) and Bombyx mori (Bm , Bm ) has been addressed through mixed-species tree construction. All four groups cluster separately, thus providing no direct evidence of orthologous sequences. However, the occurrence of gene conversion could have eliminated such evidence. The relationship between the chorion gene tree and the species cladogenic event is discussed, as are biases in codon usage, base composition, and nucleotide transformations.Correspondence to: J.C. Regier     

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.     

A complete sequence of the rice sucrose synthase-1 (RSs1) gene

Using a fragment of the maize sucrose synthase gene Sh-1 as probe, the rice genome was shown to contain at least three genes encoding sucrose synthase. One of these genes was isolated from a genomic library, and its full sequence, including 1.7 kb of 5 flanking sequence and 0.9 kb of 3 flanking sequence, is reported. The new rice gene, designated RSs1, is highly homologous to maize Sh-1 (approx. 94% identity in derived amino acid sequence), and contains an identical intron-exon structure (16 exons and 15 introns). Both RSs1 and maize Sh-1 show similar sequence homologies to a second rice sucrose synthase gene described recently (designated RSs2, Yu et al. (1992) Plant Mol Biol 18: 139–142), although both the rice genes predict an extra 6 amino acids at the C-terminus of the protein when compared to the maize gene. The RSs1 5 flanking sequence contains a number of promoter-like sequences, including putative protein-binding regions similar to maize zein genes.     

The legumin gene family: structure and evolutionary implications of Vicia faba B-type genes and pseudogenes

We have characterized several Vicia faba genes encoding methionine residue-free group B subunits of the 11S or legumin storage proteins. The respective gene subfamily consists of 10 to 15 members, six of them having been studied by DNA sequence analysis. Four functional genes (LeB2, LeB4, LeB6, LeB7) are highly homologous in their coding region and 0.3 kb of their 3 flanking sequences. On the other hand, two pseudogenes (LeB1, LeB5) have accumulated a large number of mutations including an identical 0.7 kb internal deletion; they are both flanked by a repetitive element. Analysis of sequence changes show that transitions are nearly double as frequent as transversions. CpG is the most infrequent dinucleotide whereas TpA is significantly underrepresented in exon sequences. End points of deletions are correlated with short direct repeats and preferentially found in the two introns. Our studies indicate that the Vicia faba legumin B gene subfamily contains a group of expressed, highly homologous genes as well as more diverged pseudogenes.     

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     

Three extra copies of a C4-related gene in H-2 w7 mice are C4/Slp hybrid genes generated by multiple recombinational events

Mice bearing the H-2 _w7 haplotype have five C4-related genes and constitutively express the Slp antigen. To understand the structure and evolution of the five C4-related genes of the C3H.W7 mouse, we have determined nucleotide sequences of the 5 end region of these genes. A C4/Slp hybrid nature was confirmed for three of five C4-related genes as predicted previously by restriction enzyme analysis. The nucleotide sequences of the 5 flanking regions of these three hybrid genes showed close similarity to that of the C4 gene, while the 3 side of the ninth exon of the three hybrid genes showed close similarity to that of the Slp gene. In contrast, the regions between the first exon and the middle of the ninth exon of the three hybrid genes showed a mosaic structure of C4-like and Slp-like sequences. Moreover, the boundaries of the C4-like and Slp-like sequences were quite different among the three hybrid genes. The pattern of nucleotide sequence diversity in this region among the five C4-related sequences could be mainly explained not by point mutations but by gene conversions or unequal crossovers. These results suggest that multiple genetic recombinational events between two homologous sequences played an important role in the generation and diversification of the extra copies of the C4/Slp gene in the H-2 ^w7 mouse.The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and have been assigned the accession numbers D90167-71.     

The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles

Genomic clones encoding the S ₂- and S ₆-RNases of Nicotiana alata Link and Otto, which are the allelic stylar products of the self-incompatibility (S) locus, were isolated and sequenced. Analysis of genomic DNA by pulsed-field gel electrophoresis and Southern blotting indicates the presence of only a single S-RNase gene in the N. alata genome. The sequences of the open-reading frames in the genomic and corresponding cDNA clones were identical. The organization of the genes was similar to that of other S-RNase genes from solanaceous plants. No sequence similarity was found between the DNA flanking the S ₂- and S ₆-RNase genes, despite extensive similarities between the coding regions. The DNA flanking the S ₆-RNase gene contained sequences that were moderately abundant in the genome. These repeat sequences are also present in other members of the Nicotianae.     

The chorion genes of the medfly. II. DNA sequence evolution of the autosomal chorion genes s18, s15, s19 and s16 in Diptera

We present a total of approximately 15 kb of DNA sequences, encompassing four chorion genes Ccs18, Ccs15, Ccs19, Cc16 and their flanking DNA in the medfly C. capitata. Comparison of coding regions, introns and intergenic sequences in five Dipteran species, D. melanogaster, D. subobscura, D. virilis, D. grimshawi and C. capitata documented an extensive divergence in introns and coding regions, but few well conserved elements in the proximal 5′ flanking regions in all species. These elements are related to conserved regulatory features of three of the genes, including tissue- and temporal regulation. In the fourth, gene s15, significant alterations in the 5′ flanking region may be responsible for its changed temporal regulation in C. capitata. One long intergenic sequence, located in the distal 5′ flanking region of gene s18, is homologous to ACE3, a major amplification control element and contains an 80-bp A/T-rich sequence, known to stimulate strong binding of the origin recognition complex (ORC) in D. melanogaster. Analysis of the nucleotide composition of all chorion genes in C. capitata and D. melanogaster showed that C. capitata exhibit less biased representation of synonymous codons than does D. melanogaster.     

Nucleotide sequence of a gene from Arabidopsis thaliana encoding a myb homologue

A gene encoding a proto-oncogene, a myb-related gene named Atmyb1, was cloned from Arabidopsis thaliana, and its nucleotide sequence was determined. The Atmyb1 gene contains an intron of 494 bp, and there are no highly homologous sequences present in the A. thaliana genome, but evidence was found that other myb-related genes exist. In the 5 flanking region, we found several typical cis-acting elements found in plant promoters. Sequence comparisons revealed that the ATMYB1 protein has a putative DNA-binding domain with two repeats of tryptophan clusters, which is common in MYB-related proteins in plants, while animal MYB-related proteins contain DNA-binding domains with three repeats of tryptophan clusters. The putative DNA-binding domain of the ATMYB1 protein has higher homology with that of the human c-MYB protein than with those of other plant MYB proteins.     

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA

Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNA^Val(GAC), tRNA^Asn(GUU), tRNA^Arg(ACG), tRNA^Leu(CAA), tRNA^Tyr(GUA), tRNA^Glu(UUC), tRNA^His(GUG), and tRNA^Arg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNA^Gly (UCC) and tRNA^Ile(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5 flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNA^Gly(UCC), and the tRNA^Glu(UUC) contains GATTC in its T-loop.