Partial characterization of bovine elastin gene; comparison with the gene for human elastin

A 14 kilobase (kb) genomic clone of the gene for bovine elastin, containing exons 1 and 2, has been characterized. This clone extends about 6.5 kb in the 5' direction from the initiation codon and 978 nucleotides in the 3' direction from exon 2. The size of the first intron is about 6.4 kb. The sequence immediately 5' to the initiation codon is highly conserved between the genes for bovine and human elastins and contains a TATA box consensus sequence (ATAAA), CAAT, and Sp1 binding sites. Several putative AP-2 binding sites are also present. Comparative analysis of the sequences flanking the first exon in the genes for bovine and human elastins identified conserved sequences that may be regulatory control elements. A putative enhancer core sequence is present in the first intron of the genes for bovine and human elastins.     

Molecular characterization of a sodium channel gene from the Silkworm Bombyx mori

The voltage-gated sodium channel mediates the rapid rising phase of action potentials in almost all excitable cells and is a molecular target of a variety of neurotoxins including pyrethroid insecticides. Most studies have focused on the expression of sodium channel genes in the adult stage, information on other developmental stages, however, is limited. In this study, we characterized the para sodium channel orthologous gene (BmNa(v)) of the silkworm Bombyx mori, a model insect of Lepidopteran species. The BmNa(v) gene covers a 31 kb genome region and contains 36 exons. The longest ORF contained 6258 bp and encoded 2085 amino acid residues, which shares 74%, and 77% overall amino acid sequence identities with the sodium channel proteins from Drosophila melanogaster and Blattella germanica, respectively. Using high-throughput Solexa sequence technology we conducted sequence analysis of BmNa(v) cDNAs from embryos, larvae, pupae and adults of the silkworm, identified alternative splicing sites and determined the frequencies of these splicing events in four developmental stages. Three optional exons, two sets of mutually exclusive exons, and one internal spliced exon were identified. One optional exon is unique to BmNa(v), while the others are conserved in other insect sodium channel genes. Interestingly, the expression of the mutually exclusive exons is developmentally regulated.     

Identification and characterization of a TAP-family gene in the lamprey

An expressed sequence tag obtained from a sea lamprey ( Petromyzon marinus) cDNA library was used to obtain a full-length coding sequence showing significant similarity to ABCB transporter proteins. The sequence is closely related to the mammalian ABCB9 protein and the TAP1 and TAP2 proteins that transport peptides for loading onto nascent Mhc class I molecules. The Pema-ABCB9 gene has an exon-intron organization similar to that of the mammalian TAP genes, with the exception of exon 2, which in the lamprey is split into two by a 949-bp long intron. The gene probably occurs in a single copy in the haploid lamprey genome. The ABCB9 genes appear to be evolving four-to-ten times slower than the TAP1 and TAP2 genes. Six putative transmembrane helices and the nucleotide-binding domain of the lamprey ABCB9 protein show high sequence similarity with the TAP1 and TAP2 molecules. The lamprey protein also contains sequence stretches that resemble the putative peptide interacting parts of the TAP1 and TAP2 molecules, but are peppered with ABCB9-specific residues.     

Statistical analysis of the exon-intron structure of higher eukaryote genes]

The exon-intron structure of human, insect (Drosophila sp.), and dicot plant (Arabidopsis thaliana) genes was considered. In each genome there exists a characteristic intron length. Anomalously long introns was usually the first introns in genes. In each sample there are correlations between the lengths of neighboring exons and between exon lengths and closeness to the consensus of the sites at exon boundaries. Exons and exon pairs containing an integer number of triplets are preferred. These results are relevant to the study of splicing mechanism and evolution of introns, as well as construction of gene recognition algorithms.     

Isolation and characterization of the mouse CD8 beta-chain (Ly-3) genes. Absence of an intervening sequence between V- and J-like gene segments

We have isolated and determined the nucleotide sequence and genomic organization of the genes encoding Ly-3.1 and Ly-3.2. These genes span approximately 14 kb on chromosome 6 and consist of six exons and five introns. The exons correlate roughly with the putative functional domains, namely, a leader exon, a variable and joining region-like exon, a hinge region-like exon, a transmembrane exon, and two intracytoplasmic exons. There is no intervening sequence between V- and J-like gene segments, indicating that rearrangement is not necessary for the expression of the Ly-3 gene. In the 5'-flanking region there is no "TATA" box nor "CAAT" box; however, three "GC" boxes are located upstream of the ATG initiator codon. There are short stretches of sequence homologous to 5'-flanking sequences of the Ly-2 gene. In addition, the sequences CTCTGTGGCA at -748 exhibits homology to the enhancer core sequence of the human Ig H chain and TCR genes. Comparison of the nucleotide sequence corresponding to the extracellular portion between Ly-3.1 and Ly-3.2 revealed a single base difference which results in an amino acid substitution. Therefore it is likely that this amino acid difference is responsible for the previously defined Ly-3 allotypes.     

Origin of new genes and source for N-terminal domain of the chimerical gene, jingwei, in Drosophila

This paper deals with a general question posed by the origin of new processed chimerical genes: when a new retrosequence inserts into a new genome position, how does it become activated and acquire novel protein function by recruiting new functional domains and regulatory elements? Jingwei (jgw), a newly evolved functional gene with a chimerical structure in Drosophila, provides an opportunity to examine such questions. The source of its exon encoding C-terminal peptide has been identified as an Adh retrosequence, which extends the concept of exon shuffling from recombination to retroposition as a general molecular mechanism for the origin of a new gene. However, the origin of 5' exons remains unclear. We examined two hypotheses concerning the origin of these non-Adh-derived jgw exons: (i) these exons might originate from a unique genomic sequence that fortuitously evolved a standard intron-exon structure and regulatory sequence for jgw; (ii) these exons might be a duplicate of an unrelated previously existing gene. Genomic Southern analysis, in conjunction with construction and screening of a genomic bookshelf (sub-library), was conducted in a group of Drosophila species. The results demonstrated that there are duplicate genes containing the same structure as the recruited portion of jgw. We name this duplicate gene in Drosophila teissieri and Drosophila yakuba and its orthologous gene in Drosophila melanogaster as yellow-emperor (ymp). Thus, the 5' exons/introns originated from a previously existing gene that provided new modules with specific sub-function to create jgw.     

Structure and evolution of RIM-BP genes: identification of a novel family member

RIM-binding proteins (RIM-BPs) were identified as binding partners of the presynaptic active zone proteins RIMs as well as for voltage-gated Ca(2+)-channels. They were suggested to form a functional link between the synaptic-vesicle fusion apparatus and Ca(2+)-channels. Here we show that the RIM-BP gene family diversified in different stages during evolution, but retained their unique domain structure. While invertebrate genomes contain one, and vertebrates include at least two RIM-BPs, we identified an additional gene, RIM-BP3, which is exclusively expressed in mammals. RIM-BP3 is encoded by a single exon of which three copies are present in the human genome. All RIM-BP genes encode proteins with three SH3-domains and two to three fibronectin III repeats. The flanking regions diverge in size and sequence and are alternatively spliced in RIM-BP1 and -2. Quantitative real-time RT-PCR and in situ hybridization analyses revealed overlapping but distinct expression patterns throughout the brain for RIM-BP1 and -2, while RIM-BP3 was detected at high levels outside the nervous system. The modular domain structure of RIM-BPs, their expression pattern and the conservative expansion during evolution shown here support their potential role as important molecular adaptors.     

Molecular genetics of human erythrocyte MiIII and MiVI glycophorins. Use of a pseudoexon in construction of two delta-alpha-delta hybrid genes resulting in antigenic diversification

Human glycophorins alpha and delta (or A and B) specify the MNSs blood group antigens; they exhibit considerable structural variation among populations. We show that two variant phenotypes of Miltenberger class III and VI are encoded by similar hybrid glycophorin genes in a delta-alpha-delta arrangement. Restriction mapping identified altered fragments unique to the MiIII and MiVI genes. Genomic sequences spanning exons 2 to 4 of the two genes were obtained by allele-specific polymerase chain reaction. Restriction analysis and direct sequencing of the amplified DNA revealed that MiIII and MiVI genes are identical to the delta gene except that, in both, an internal segment of the delta gene has been replaced by its homologous counterpart of the alpha gene, resulting in a delta-alpha-delta hybrid structure. In the process of hybrid formation a portion of alpha exon 3 and intron 3, that carries a functional 5' splicing signal, has been fused to an exon-like sequence in the delta gene that retains a 3' but lacks a 5' splicing signal. These rearrangements created a composite exon resulting in the expression of the ordinarily unexpressed delta gene sequence and conferred the hybrid proteins with new antigenic specificities. The expression of this sequence in MiIII glycophorin is directly demonstrated by protein sequencing. MiIII and MiVI genes differ in the location of upstream (delta-alpha) and downstream (alpha-delta) breakpoints and in the length of sequence replacement. The delta-alpha breakpoints of the two genes occur at different locations within a 35-base pair sequence of exon 3 that is clustered with multiple inverted repeats, whereas the alpha-delta breakpoints reside downstream in two dissimilar blocks of sequences of intron 3. The minimal length of the delta gene sequence that has been replaced by the alpha gene is 55 base pairs in the MiIII gene and 131 base pairs in the MiVI gene. Such segmental DNA transfers may have proceeded unidirectionally through the mechanisms of gene conversion.     

Structure and regulation of the envoplakin gene

Envoplakin, a member of the plakin family of proteins, is a component of desmosomes and the epidermal cornified envelope. To understand how envoplakin expression is regulated, we have analyzed the structure of the mouse envoplakin gene and characterized the promoters of both the human and mouse genes. The mouse gene consists of 22 exons and maps to chromosome 11E1, syntenic to the location of the human gene on 17q25. The exon-intron structure of the mouse envoplakin gene is common to all members of the plakin family: the N-terminal protein domain is encoded by 21 small exons, and the central rod domain and the C-terminal globular domain are coded by a single large exon. The C terminus shows the highest sequence conservation between mouse and human envoplakins and between envoplakin and the other family members. The N terminus is also conserved, with sequence homology extending to Drosophila Kakapo. A region between nucleotides -101 and 288 was necessary for promoter activity in transiently transfected primary keratinocytes. This region is highly conserved between the human and mouse genes and contains at least two different positively acting elements identified by site-directed mutagenesis and electrophoretic mobility shift assays. Mutation of a GC box binding Sp1 and Sp3 proteins or a combined E box and Krüppel-like element interacting with unidentified nuclear proteins virtually abolished promoter activity. 600 base pairs of the mouse upstream sequence was sufficient to drive expression of a beta-galactosidase reporter gene in the suprabasal layers of epidermis, esophagus, and forestomach of transgenic mice. Thus, we have identified a regulatory region in the envoplakin gene that can account for the expression pattern of the endogenous protein in stratified squamous epithelia.     

Comparative sequence analysis of equine and human MHC class II DQB genes

The MHC class II DQB gene of horse was isolated and characterized. No obvious mutations causing frame shifts, or destruction of putative protein structure and splicing machinery were detected. Nucleotide sequence of exon 2 was consistent with an allelic sequence of the W23 haplotype. The cytoplasmic region of the equine DQB gene comprised two exons and an intron. A novel fragment of the gene was identified at the 3' intergenic region proximal to the ELA-DQB gene by sequence comparison between the human and horse DQB genes. This sequence showed the highest identity to exon 3 region of the DQB gene, however the 5' half of this exon was truncated as compared with the intact exon. This gene fragment was also identified in the same site of the HLA-DQB gene.