Structure and evolution of the bovine prothrombin gene

The cloned bovine prothrombin gene has been characterized by partial DNA sequence analysis, including the 5' and 3' flanking sequences and all the intron-exon junctions. The gene is approximately 15.4 x 10(3) base-pairs in length and comprises 14 exons interrupted by 13 introns. The exons coding for the prepro-leader peptide and the gamma-carboxyglutamic acid-containing region are similar in organization to the corresponding exons in the factor IX and protein C genes. This region has probably evolved as a result of recent gene duplication and exon shuffling events. The exons coding for the kringles and the serine protease region of the prothrombin gene are different in organization from the homologous regions in other genes, suggesting that introns have been inserted into these regions after the initial gene duplication events.     

Analysis of mouse major urinary protein genes: variation between the exonic sequences of group 1 genes and a comparison with an active gene out with group 1 both suggest that gene conversion has occurred between MUP genes

Here we compare the exonic sequences of four Group 1 mouse major urinary protein (MUP) genes and four Group 1 cDNA sequences. These define seven different nucleotide sequences which differ from each other by 0.35% of bases on average, and which would code for seven different MUP proteins that could probably be resolved physically into at least five classes. The sequences differ at 13 nucleotide positions and at six codons, and although they are closely related their descent cannot be described by a simple series of duplications. We also describe the sequence of another liver cDNA (pMUP15) which has diverged from the Group 1 consensus sequence in 14.6% of bases. The divergence is much greater over exons 1-3 than over exons 4-6, suggesting that an ancestral gene conversion event has occurred. pMUP15 also differs from the Group 1 genes in having a longer signal peptide sequence and a different splice configuration between exons 6 and 7. Unlike the Group 1 sequences, pMUP15 contains a potential N-linked glycosylation site. Other published work has shown that a shorter cDNA clone which is identical over their common sequence to pMUP15 codes for MUP proteins that are unusually large in size and acidic in pI. We show here that mouse urine does indeed contain a glycosylated MUP protein with those properties, presumably the product of the gene that corresponds to pMUP15.     

Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida

Chalcone synthase-encoding genes (chs) in Petunia hybrida comprise a multigene family. Some of the chs genes have been grouped into a subfamily, based upon their strong cross-hybridization and tight genomic linkage. From genomic libraries eight 'complete' chs genes, two chs gene 5'-fragments and two chs gene 3'-fragments have been isolated. The nucleotide sequence of six complete chs genes is presented and discussed in relation to their evolutionary origin and expression in different tissues. Each member of the family consists of two exons separated by an intron of variable size and sequence, which is located at a conserved position. The chs gene fragments represent single exons. Homology between non-linked chs genes is approx. 80% at the DNA level and restricted to protein-coding sequences. Homology between subfamily members (which are tightly linked) is higher (90-99%) and extends into untranslated regions of the gene, strengthening the view that they arose by recent gene duplications. The chsD gene contains a mutated translation stop codon, suggesting that this is an inactive (pseudo)gene. None of the other members of the gene family exhibits characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must have been a recent event. Homology at the protein level between some (expressed) chs genes is surprisingly low. The possibility that these genes encode proteins with slightly different enzymatic activities is discussed.     

Molecular biology of intracellular protein trafficking

The evidence accumulated to date indicates that protein compartmentalization is mediated through specific regions of proteins destined for translocation into subcellular organelles. Proteins targeted to mitochondria, chloroplasts or the endoplasmic reticulum have 'transit' sequences contained in amino-terminal peptide extensions. However, most peroxisomal proteins do not have amino-terminal extensions. Protein importation into mitochondria has been extensively studied and characterized. This post-translational process appears to involve receptors on the mitochondrial outer membrane, and is dependent upon the electrochemical gradient across the inner membrane. Translocation to one of the submitochondrial compartments is determined by the type of transit sequence contained in a mitochondrial protein. The majority of imported mitochondrial proteins are proteolytically altered prior to assembly into oligomeric enzyme complexes. Protein importation into peroxisomes is distinctly different from importation into mitochondria. Although both processes are post-translational, their only other similarity is a requirement for ATP. In this review, we present and compare recent evidence for both mitochondrial and peroxisomal protein importation.     

Heterogeneity in the cysteine protease inhibitor clitocypin gene family

Clitocypin from the basidiomycete Clitocybe nebularis is the first fungal protein cysteine protease inhibitor to be characterised in detail, yet no information on its molecular genetics is available. Owing to its unique characteristics, it was assigned as the only member of a new family of cysteine protease inhibitors in the MEROPS inhibitor classification. Here we describe the full-length sequence of the clitocypin gene. A BLAST search confirmed its lack of significant sequence similarity to any other gene. The gene is composed of four exons and three short introns and belongs to a small family of closely related genes with more than 90% identity. Sequence variability is evenly distributed in introns and exons and deduced amino acid substitutions are distributed throughout the protein sequence. Basidiocarps collected at two distant locations were examined and the level of heterogeneity found in one basidiocarp is similar to that between the two. Sequencing of the ribosomal DNA spacers from the two basidiocarps confirmed that the heterogeneity observed in the clitocypin gene is not due to evolutionary divergence of the two specimens caused by geographic separation. Clitocypin is expressed in different parts of the basidiocarp and in cultured mycelia in a manner suggesting regulation by developmental and/or environmental factors.     

Sequences encoding two trypsin inhibitors occur in strikingly similar genomic environments.

The nucleotide sequences of two approx. 4 kilobase pair segments of the bovine genome are presented. One segment contains a coding region for bovine pancreatic trypsin inhibitor (BPTI) and the other segment contains a coding region for a BPTI homologue. The two 4 kilobase pair sequences are strikingly similar over approx. 3.4 kilobase pairs of their sequence, including putative intron sequences, suggesting that they have evolved from a gene duplication event.     

Ancestral exonic organization of FUT8, the gene encoding the alpha6-fucosyltransferase, reveals successive peptide domains which suggest a particular three-dimensional core structure for the alpha6-fucosyltransferase family

Based on PCR strategies and expression studies, we define the genomic organization of the FUT8b gene. This gene encodes the only known mammalian enzyme transferring fucose in an alpha1-->6 linkage on the asparagine-branched GlcNAc residue of the chitobiose unit of complex N:-glycans. The intron/exon organization of the bovine coding sequence determines five successive functional domains. The first exon encodes a domain homologous to cytoskeleton proteins, the second presents a proline-rich region including a motif XPXPPYXP similar to the peptide ligand of the SH3-domain proteins, the third encodes a gyrase-like domain (an enzyme which can bind nucleotides), and the fourth encodes a peptide sequence homologous to the catalytic domain of proteins transferring sugars. Finally, the last exon encodes a domain homologous to the SH3 conserved motif of the SH2-SH3 protein family. This organization suggests that intramolecular interactions might give a tulip-shaped scaffolding, including the catalytic pocket of the enzyme in the Golgi lumen. Deduced from the published sequence of chromosome 14 (AL109847), the human gene organization of FUT8 seems to be similar to that of bovine FUT8b, although the exon partition is more pronounced (bovine exons 1 and 2 correspond to human exons 1-6). The mosaicism and phylogenetic positions of the alpha6-fucosyltransferase genes are compared with those of other fucosyltransferase genes.     

Five distinct calcium and phospholipid binding proteins share homology with lipocortin I

We have purified two 35-kDa proteins from rat peritoneal lavages that inhibit phospholipase A2 activity. Both are calcium/phospholipid-dependent membrane binding proteins and share similar structural and biochemical properties with lipocortins I and II. By sequence analysis we confirmed that they are lipocortin-related, and we refer to the two inhibitors as lipocortins III and V. Using partial sequence information obtained from the purified rat proteins, full length cDNA clones for both proteins and for their human counterparts were isolated. As with lipocortins I and II, the amino acid sequences of lipocortins III and V which were deduced from the cDNA clones are highly conserved, sharing 50% identity with other family members. Related proteins were also purified from bovine intestinal mucosa and characterized by peptide mapping, sequence, and immunological analyses. In addition to lipocortins III and V the bovine preparation contained a third 35-kDa inhibitor and a 68-kDa inhibitor, extending the number of known lipocortins to six distinct proteins. While the various lipocortins are structurally similar, distinct differences in their cellular distribution indicate specialized roles for the individual proteins.     

Is there VDAC in cell compartments other than the mitochondria?

Higher eukaryotes, including mammals and plants, express a family of VDAC proteins each encoded by a distinct gene. Two human genes encoding VDAC isoforms (HVDAC1 and HVDAC2) have been characterized in greatest detail. These genes generate three proteins that differ primarily by the addition of distinct N terminal extensions in HVDAC2 and HVDAC2, a splice variant of HVDAC2, relative to HVDAC1. Since N terminal sequences have been demonstrated to target many proteins to appropriate subcellular compartments, this observation raises the possibility that the N terminal differences found in HVDAC isoforms may lead to targeting of each protein to different cellular locations. Consistent with this hypothesis, a large number of reports have provided evidence consistent with the notion that HVDAC1 and its homolog in related mammalian species may specifically be present in the plasma membrane or other nonmitochondrial cellular compartments. Here, we review this information and conclude that if VDAC molecules are present at nonmitochondrial locations in mammalian cells, these are unlikely to be the known products of the HVDAC1 or HVDAC2 genes.     

Organization of the human gene encoding the epididymis-specific EP2 protein variants and its relationship to defensin genes

The EP2 gene codes for at least nine message variants that are all specifically expressed in the epididymis. These variants putatively encode small secretory proteins that differ in their N- and C-termini, resulting in proteins that can have little or no sequence similarity to each other. We have isolated and sequenced the human EP2 gene to determine the molecular origin of these variants. The EP2 gene has two promoters, eight exons, and seven introns. Exons 3 and 6 encode protein sequences homologous to beta-defensins, a family of antimicrobial peptides. This sequence homology and the arrangement of promoters and defensin-encoding exons suggest that the EP2 gene originated from two ancestral beta-defensin genes arranged in tandem, each contributing a promoter and two exons encoding a leader sequence and a defensin peptide. The proposed evolutionary relationship between the EP2 gene and defensin genes is supported by the observation that the EP2 gene is located on chromosome 8p23 near the defensin gene cluster and is separated by 100 kilobases or less from DEFB2, the gene for beta-defensin-2. While the EP2 gene transcribes beta-defensin-like message variants, most of the known message variants code for nondefensin proteins or proteins containing only a partial defensin peptide sequence. We suggest that, during its evolution, the EP2 gene has acquired new functions that may be important for sperm maturation and/or storage in the epididymis.