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.     

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.     

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

An evolutionary model for the duplication and divergence of esterase genes in Drosophila

Summary The esterase 5 (Est-5 = gene, EST 5 = protein) enzyme in Drosophila pseudoobscura is encoded by one of three paralogous genes, Est-5A, Est5B, and Est-5C, that are tightly clustered on the right arm of the X chromosome. The homologous Est-6 locus in Drosophila melanogaster has only one paralogous neighbor, Est-P. Comparisons of coding and flanking DNA sequences among the three D. pseudoobscura and two D. melanogaster genes suggest that two paralogous genes were present before the divergence of D. pseudoobscura from D. melanogaster and that, later, a second duplication occurred in D. pseudoobscura. Nucleotide sequences of the coding regions of the three D. pseudoobscura genes showed 78–85% similarity in pairwise comparisons, whereas the relatedness between Est-6 and Est-P was only 67%. The higher degree of conservation in D. pseudoobscura likely results from the comparatively recent divergence of Est-5B and Est-5C and from possible gene conversion events between Est-5A and Est-5B. Analyses of silent and replacement site differences in the two exons of the paralogous and orthologous genes in each species indicate that common selective forces are acting on all five loci. Further evidence for common purifying selective constraints comes from the conservation of hydropathy profiles and proposed catalytic residues. However, different levels of amino acid substitution between the paralogous genes in D. melanogaster relative to those in D. pseudoobscura suggest that interspecific differences in selection also exist.Offprint requests to: R.C. Richmond     

Cloning and sequencing of complement component C9 and its linkage to DOC-2 in the pufferfish Fugu rubripes

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6–7 kb from C9 in a head-to-head or 5′ to 5′ orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.     

Similar tissue-specific expression of the Adh genes from different Drosophila species is mediated by distinct arrangements of cis-acting sequences

In Drosophila melanogaster transformants, the alcohol dehydrogenase (Adh) genes from D. affinidisjuncta and D. grimshawi show similar levels of expression except in the adult midgut where the D. affinidisjuncta gene is expressed about 10- to 20-fold more strongly. To study the arrangement of cis-acting sequences responsible for this regulatory difference, homologous restriction sites were used to create a series of chimeric genes that switched fragments from the 5 and 3 flanking regions of these two genes. Chimeric genes were introduced into the germ-line of D. melanogaster, and Adh gene expression was analyzed by measuring RNA levels. Various gene fragments in the promoter region and elsewhere influence expression in the adult midgut and in whole larvae and adults. Comparison of these results with earlier studies involving chimeras between the D. affinidisjuncta and D. hawaiiensis genes indicates that expression in the adult midgut is influenced by multiple regulatory sequences and that distinct arrangements of regulatory sequences can result in similar levels of expression both in the adult midgut and in the whole organism.     

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     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

De novo origin of new genes with introns in Plasmodium vivax

The origin of new genes is critical for organisms adapting to new niches. Here, we present evidence for a recent de novo origin of at least 13 protein-coding genes in the genome of Plasmodium vivax. Although recently de novo originated genes have often been suggested to be initially intronless, five of the genes identified in our analysis contain introns in their coding regions. Further investigations revealed that these introns likely evolved from previously intergenic regions together with the coding sequences. We discuss the potential mechanisms for intron formation in these genes and propose that intronization be considered in the formation of de novo originated genes.     

Organization and expression of mouse Hox3 cluster genes

Summary The three yolk protein genes (yp) of Drosophila melanogaster are transcribed in a sex- and tissue-limited fashion. We have searched for cis-regulatory sequences in regions flanking yp1 and yp2 to identify the elements that confer female-specific expression in the fat body. One such 127 by element has previously been identified in this region. We show here the existence of two additional regions which confer female fat body-specific expression on an Adh reporter gene and on the native yp2 gene, respectively. This suggests some redundancy in the regulation of expression of the yp genes. Computer searches for putative binding sites for the DSX protein, which regulates sex-specific expression of the yp genes, revealed several such sites in our constructs. However, the significance of these is unclear since many such sites also occur in genes which one would not expect to be regulated in a sex-specific manner (e.g. Adh, Actin 5C). We suggest that DSX acts in concert with other proteins to mediate sex- and tissue-specific expression of the yp genes.     

Cotranscriptional expression of mitochondrial genes for subunits of NADH dehydrogenase, nad5, nad4, nad2, in Marchantia polymorpha

Three genes for the subunits of the NADH dehydrogenase (nad5, nad4, and nad2) are tandemly clustered on the liverwort mitochondrial genome. Their gene products showed high levels of amino acid sequence identity with the corresponding subunits from higher plant mitochondria (82.8–84.4%), and significant levels of identity with those from liverwort chloroplast (32.0–33.5%), Podospora anserina mitochondria (21.4–45.9%), and human mitochondria (18.4–27.9%). In addition, these three subunits from liverwort mitochondria have conserved amino acid residues in their central regions. The gene nad5 is interrupted by a 672 by group I intron, while genes nad4 and nad2 are interrupted by group II introns of 899 by and 1418 bp, respectively. Northern blot analysis using exon-intron specific probes indicated that these three genes are transcribed as a single precursor mRNA of 9.6 kb in length and are processed into mature mRNA molecules in liverwort mitochondria. Several regions of this nad gene cluster are repeated in the liverwort mitochondrial genome.Communicated by R.G. Herrmann     

Allelic variation at the nucleotide level in Drosophila glue genes

Summary We have sequenced a 4.7 kb genomic fragment from the chromosomal region 68C of the Drosophila melanogaster wild-type strain Formosa which contains the salivary gland secretion protein genes sgs3 and sgs7. A comparison of this sequence with that from the same region of Oregon R (Garfinkel et al. 1983) shows that the major difference in this segment is a 300 bp region, corresponding to 20 of the 15 bp tandemly repeated units which compose the central coding region of sgs3 Oregon, which is absent in the Formosa strain. The order of the remaining repeated units, common to the two strains, suggests that this region is relatively stable and does not undergo frequent rearrangements or mutations. The 5 and 3 flanking sequences of the transcribed regions, which represent ca. seventy percent of our sequencing data, appear to be as strongly conserved as the transcribed sequences themselves. In the course of in vitro manipulations of the Formosa sequence, we have isolated two spontaneous deletions of repeated units in the coding region of sgs3 and discuss possible mechanisms leading to variation in sgs3 alleles.     

Cloning and characterization of chloroplast ribosomal protein-encoding genes, rpl16 and rps3, of the marine macro-algae, Gracilaria tenuistipitata

In Gracilaria tenuistipitata, a highly differentiated multicellular member of the marine red algae, Rhodophyta, chloroplast (cp) DNA can be separated as a satellite band from the nuclear DNA in a CsCl gradient. Using a heterologous probe from Chlamydomonas, the ribosomal protein-encoding gene, rpl16, was located on a 4.5-kb EcoRI fragment of cp DNA. The fragment was cloned and a 1365-bp region around rpl16 was sequenced. The gene order around rpl16, 5′ rpl22-rps3-rpl16, is identical to that detected in the chloroplast DNA of liverwort, tobacco and maize. Both the nucleotide sequence and the amino-acid sequence of rpl16 are more conserved than that of rps3. The rpl16 gene contains no intron, a feature which shows more similarity to the unicellular green algae, Chlamydomonas, than the other land plants. Sequences that may form a stable stem-loop structure were detected within the coding sequence of rpl16.     

Phylogenetics of early branching eudicots:Comparing phylogenetic signal across plastid introns,spacers,and genes

<正>Recent phylogenetic analyses revealed a grade with Ranunculales,Sabiales,Proteales,Trochodendrales, and Buxales as first branching eudicots,with the respective positions of Proteales and Sabiales still lacking statistical confidence.As previous analyses of conserved plastid genes remain inconclusive,we aimed to use and evaluate a representative set of plastid introns(group I:trnL;group II:petD,rpll6,trnK) and intergenic spacers(trnL-F,petB-petD, atpB-rbcL,rps3-rpl16) in comparison to the rapidly evolving matK and slowly evolving atpB and rbcL genes. Overall patterns of micro structural mutations converged across genomic regions,underscoring the existence of a general mutational pattern throughout the plastid genome.Phylogenetic signal differed strongly between functionally and structurally different genomic regions and was highest in matK,followed by spacers,then group II and group I introns.The more conserved atpB and rbcL coding regions showed distinctly lower phylogenetic information content.Parsimony,maximum likelihood,and Bayesian phylogenetic analyses based on the combined dataset of non-coding and rapidly evolving regions(14 000 aligned characters) converged to a backbone topology of eudicots with Ranunculales branching first,a Proteales-Sabiales clade second,followed by Trochodendrales and Buxales. Gunnerales generally appeared as sister to all remaining core eudicots with maximum support.Our results show that a small number of intron and spacer sequences allow similar insights into phylogenetic relationships of eudicots compared to datasets of many combined genes.The non-coding proportion of the plastid genome thus can be considered an important information source for plastid phylogenomics.     

Characterization of the 5′-to-5′linked adult α- and β-globin genes from three sciaenid fish species (Pseudosciaena crocea, Sciaenops ocellatus, Nibea miichthioides)

Recently, we cloned the adult α-globin genes from large yellow croaker Pseudosciaena crocea, cuneate drum Nibea miichthioides and red drum Sciaenops ocellatus. All these α-globins have a unique Gly insertion at the 47th residue. In this paper, the three sciaenid globin complexes were identified and compared in detail. Linkage analysis indicated that the sciaenid α- and β-globin genes were oriented head-to-head relative to each other. The sciaenid intergenic regions between the linked α- and β-globin genes were the smallest in reported fish globin gene complexes to date. Classical promoter elements were condensed and the CCAAT box unstable duplication was found in these regions. The promoter function of the intergenic region from large yellow croaker was tested by transient expression of EGFP in Vero cells. We also described a method for studying luciferase reporter gene transient expression in primary fish erythrocytes. We used the method to assess the promoter strength of the three intergenic regions between the sciaenid α- and β-globin genes.     

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum, a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

Structural and genetic studies of the proliferation disrupter genes of Drosophila simulans and D. melanogaster

To examine whether structural and functional differences exist in the proliferation disrupter (prod) genes between Drosophila simulans and D. melanogaster, we analyzed and compared both genes. The exon–intron structure of the genes was found to be the same – three exons were interrupted by two introns, although a previous report suggested that only one intron existed in D. melanogaster. The prod genes of D. simulans and D. melanogaster both turn out to encode 346 amino acids, not 301 as previously reported for D. melanogaster. The numbers of nucleotide substitutions in the prod genes was 0.0747 ±  per synonymous site and 0.0116 ± 0.0039 per replacement site, both comparable to those previously known for homologous genes between D. simulans and D. melanogaster. Genetic analysis demonstrated that D. simulans PROD can compensate for a deficiency of D. melanogaster PROD in hybrids. The PRODs of D. simulans and D. melanogaster presumably share the same function and a conserved working mechanism. The prod gene showed no significant interaction with the lethality of the male hybrid between these species. This revised version was published online in July 2006 with corrections to the Cover Date.