Carbonic Anhydrase-Related Protein XI: Structure of the Gene in the Greater False Vampire Bat (Megaderma lyra) Compared with Human and Domestic Pig

Carbonic anhydrase-related protein XI (CA-RP XI) is a member of the α-carbonic anhydrase family (encoded by the gene CA-11), which has lost features of the active site required for enzymatic activity. Using PCR, we amplified CA-11 from genomic DNA of the bat Megaderma lyra. To elucidate the gene structure, we sequenced PCR products and compared their sequences with genomic and mRNA sequences known from human and domestic pig. We identified and sequenced eight introns in the bat CA-11. Five introns (introns 3–7) are located in identical or similar positions in other members of the vertebrate α-carbonic anhydrase gene family. Two 5′ introns and one 3′ intron are located in the regions of little or no sequence similarity with other members of the gene family. The low sequence similarity and additional introns suggest a separate evolutionary origin for the 5′ and 3′ portions of the CA-RP XI gene.     

A complex glutathione transferase gene family in the housefly Musca domestica

In most metazoans, the glutathione S-transferases (GST) are encoded by gene families, and are used to detoxify xenobiotics. We describe the structure of genomic loci coding for the GSTs in the housefly that have been implicated, by both genetic and biochemical means, in mediating insecticide resistance. In earlier work, we showed that one of the theta-class enzymes, MdGST-3, is overproduced in resistant flies and degrades certain insecticides. We used a fragment from a cDNA clone of MdGST-3 as a probe to screen a housefly genomic DNA bank in phage λ. This probe detected multiple gst loci. Genes for GSTs were found in five different, nonoverlapping λ clones, three of which carry multiple, closely linked gsts. Multiple genes for both MdGST-3 and MdGST-4 were found; some of which have introns in their 5′ untranslated regions. In adults, the only MdGST-3 enzymes that are expressed are encoded by the intron-free genes. A new theta-class GST (called MdGST-5) was also discovered. Fusion genes comprising 5′ MdGST-3 sequences and either MdGST-4 or MdGST-5 sequences in their 3′ halves were encountered at three separate loci. The genes described here are found in both the ancestral sensitive strain and the insecticide-resistant strains.     

The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization,evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5′ border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3′ acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

Growth arrest of Schizosaccharomyces pombe following overexpression of mouse type 1 protein phosphatases

The rice disease resistance gene Xa21, which encodes a receptor-like kinase, is a member of a multigene family. Based on comparisons of genomic?sequences of seven family members, seventeen transposon-like elements were identified in the 5′ and 3′ flanking regions and introns of these genes. Sequence characterization revealed that these elements are diverse, showing similarity to maize Ds, CACTA and miniature inverted repeat-like elements, as well as novel elements. Only two elements were located in presumed coding regions, indicating that integration of transposable elements at the Xa21 disease resistance locus occurred preferentially in noncoding regions.     

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.     

Identification and characterization of protease inhibitors in Diplotaxis species

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.     

Sequence and expression of the discoidin I gene family in Dictyostelium discoideum

We have isolated recombinant phage and plasmids containing the four developmentally regulated discoidin I genes of Dictyostelium discoideum. Two of the genes are linked within 0.5 × 10³ bases with the same polarity. S¹ nuclease mapping shows that at least three members of the gene family are expressed and that the 5′ ends of the mRNAs start at equivalent sites. The genes have homologous 5′ untranslated regions and extremely divergent 3′ untranslated regions. In addition, some of the genes are flanked by homologous repeat sequences. The genes encode three different isoelectric forms of the protein. Examination of nucleotide sequences in the protein coding region shows that most nucleotide changes in the 5′ half of the gene result in amino acid substitutions while most base substitutions in the 3′ half are neutral.     

Sequence homologies in the mouse protamine 1 and 2 genes

To identify candidates for cis-acting sequences that regulate the stage and cell-specific expression of the two coordinately regulated protamine genes in the mouse, genomic clones were isolated and the nucleotide sequences of the 5′ flanking regions and coding regions were compared. Unlike most histone genes and the multigene family of trout protamine genes which are intronless, each mouse protamine gene has a single, short intervening sequence. Although the coding regions do not share significant nucleotide homology, the 5′ flanking regions contain several short homologous sequences that may be involved in gene regulation. An additional shared sequence is present in the 3′ untranslated region surrounding the poly(A) addition signal in both genes.     

Molecular variation of the shuttle craft and Lim3 genes, controlling the development of the nervous system, in a natural Drosophila melanogaster population

Comparative polymorphism of the first exon and first intron of the shuttle craft (stc) and Lim3 genes and their putative regulatory 5′-flanking sequences was analyzed using 20 sequenced natural alleles. A comparison of the stc and Lim3 genes showed that the extent of polymorphism was similar in their introns and corresponded to the variation level characteristic of Drosophila melanogaster, while the putative regulatory region and first intron of the stc gene proved to be more variable than the corresponding regions of the Lim3 gene. Since the genes under study occurred on the same chromosomes isolated from one population and were close together in a region having a high recombination rate, the difference in the extent of polymorphism between the regulatory and coding regions was explained by individual characteristics of each gene. The results made it possible to assume that the extent of polymorphism of the coding gene regions is maintained by balancing selection.     

Polymorphism and evolution of collagenolytic serine protease genes in crustaceans.

Two genomic DNA fragments encoding crustacean collagenolytic serine protease genes show coding fragments that span 1522-1526 base pairs and contain seven exons encoding the complete amino acid sequence of two enzymes, CHYA and CHYB. As in serine protease genes from other organisms, the region coding for the residues around the active site is split by two introns. Although the introns differ from those of other organisms in size and nucleotide sequence, their number and location are more or less the same as found in mammalian chymotrypsin or elastase genes that evolved lately, but different for trypsin genes. Meanwhile, the junction that occurs between the propeptide and the maturation site is only found in the shrimp genes. This is also the case for the junction located 13 amino acids after the active site aspartic acid in these genes. Between 40 and 50 copies of the genes are reported by Southern analysis. Seven different genes within ChyA Pv family present 0-6% base changes, whereas five different genes belonging to ChyB Pv family show changes of up to 27% in the short studied portion of exon 4. This last family presents a mosaic organization of the coding parts, which are also expressed in the hepatopancreas of the shrimp as the variant PVC5 cDNA.