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.     

Heterogeneity of amelogenin mRNA in the bovine tooth germ

The amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. To study the molecular mechanisms underlying the heterogeneity of the amelogenins we isolated cDNA clones encoding these proteins. The clones were definitively identified by hybrid-selected translation experiments and by comparison of the DNA sequence with the protein-derived amino acid sequence. Southern hybridization of bovine genomic DNA indicated that amelogenin is a single copy gene. However, Northern hybridization experiments distinctly showed two major species of mRNA, each of which were sufficiently large enough to encode the highest known molecular weight species of amelogenin proteins. Furthermore, immunoprecipitation of hybrid-selected translation products using isolated amelogenin cDNA showed multiple, translated protein products. These data are supportive of a differential mRNA processing mechanism involved in generating a heterogeneous family of amelogenin matrix proteins from a single gene.     

Human amelogenin up-regulates osteogenic gene expression in human bone marrow stroma cells

Extracts of enamel matrix proteins are used to regenerate periodontal tissues. Amelogenin, the most abundant enamel protein, plays an important role in the regeneration of these tissues. However, the molecular mechanisms by which amelogenin contributes to periodontal regeneration remain unknown. Using primary human bone marrow stroma cells (hBMSCs) transduced with lentivirus encoding human amelogenin (hAm), we performed genome-wide expression profiling to analyze the effects of hAm transduction on the regulation of genes involved in osteogenic differentiation. Our results revealed that BMP-2, BMP-6, OPN and VEGFC were up-regulated. These results suggest that hAm may be a key element in regulating hBMSCs osteogenic differentiation.     

Studies of the secondary structures of amelogenin from bovine tooth enamel

Circular dichroism and Fourier transform infrared spectroscopic studies of the major amelogenin protein of developing bovine tooth enamel in solution and in the solid state suggest a unique secondary structure containing beta-sheet and repetitive beta-turn structures. The repetitive beta-turn structure at the C-terminal end results from the unique primary structure of amelogenin.     

Structure and expression of the mouse prealbumin gene

We cloned a genomic DNA fragment which covers the entire sequence of the mouse prealbumin gene and then studied the structure. The coding regions are separated into four exons by three introns, and these numbers, the sizes of the exons and the relative sites of the exon-intron junctions are all in complete agreement with those determined for the human gene. The sequences of four exons can be aligned perfectly with that of the previously determined mouse prealbumin cDNA. In addition to the exon regions, we found two highly conserved DNA regions between the mouse and human prealbumin genes, one in the 5'-flanking region of the gene and the other in the 3' end region of the first intron. These DNA regions contain several consensus glucocorticoid receptor-binding site sequences, and the latter also contains an enhancer sequence present in the immunoglobulin kappa-chain joining-constant kappa intron. RNA hybridizing to the mouse prealbumin cDNA was detected in the extracts from liver, brain, and kidney, but was not detected in testes, spleen, or heart. Little change was caused in the level of prealbumin mRNA in the liver by administration of dexamethasone to mice.     

Complete amino acid sequence of amelogenin in developing bovine enamel

Pure amelogenin protein in developing bovine incisor enamel was isolated and its primary structure was investigated by sequencing the peptides obtained after clostripain and chymotrypsin digestions and CNBr degradation with an automated Edman sequencer. The enamel protein was found to be composed of 170 amino acid residues with one phosphate having a molecular weight of 19,350 and its complete amino acid sequence was elucidated. This protein has no sequence homology with any other tissue or secretory protein of known structure.     

Single bovine sperm sex typing by amelogenin nested PCR

Sex-sorted bovine semen has become a valuable tool in animal production for sex preselection. Development of novel sperm sexing technologies, or evaluation of the quality of existing methods, often requires a single-sperm, sex-typing method that is reliable and easy to perform. In the present study, we report the development, validation, and application of a simple, reliable, and cost-effective method for single-sperm sex typing using nested polymerase chain reaction (PCR), based on the amelogenin gene. Several hundred single sperm were isolated using a simple manual technique, or a high-speed flow-sorter, and were successfully sex-typed using the amelogenin nested PCR. Based on the pooled results of individual sperm, there was no significant difference in the semen sex ratio of unsorted (44.6% X-sperm and 55.4% Y-sperm) or X/Y-sorted semen (91.4% X-sperm and 94.0% Y-sperm), as compared to the expected ratio in unsorted semen or the post-sorting reanalysis data, respectively. The amelogenin single-sperm sexing method was an adaptable, accurate, and reliable tool for single-sperm sex typing.     

Structure and analysis of the bovine atrial natriuretic peptide precursor gene

The isolation and sequence analysis of the gene encoding the bovine atrial natriuretic peptide (ANP) precursor is described. The bovine-ANP coding sequences are located on three exons which are interrupted by two intervening sequences. Comparison of the bovine, human, rat and mouse ANP gene sequences reveals a common organization of introns and exons and a high degree of sequence homology in the 5'-flanking and coding regions. Examination of the pre-proANP amino acid sequence derived from the bovine gene with those from rat, mouse and human, indicates a high degree of sequence homology in both the amino-terminal and biologically-active carboxy-terminal ANP region. The latter region in the bovine sequence resembles its human counterpart except for a carboxy-terminal Arg-Arg dipeptide.     

A PCR-based sex-determination assay in cattle based on the bovine amelogenin locus

A method for determining the sex of bovine embryos has been established. Primers for a portion of the bovine amelogenin locus (AMX/Y) were used to amplify DNA present in either 0.1 μ1 of blood or biopsies taken from 6–7-day-old embryos. The primers amplify a 280 bp band in females and a 280 and 217bp bands in males. The method is rapid, does not require prior purification of DNA and contains an internal control which detects PCR failure.     

Structure of the 3' portion of the bovine elastin gene

A bovine genomic library constructed by partial Sau3A digestion and contained in lambda Charon 30 was screened by in situ hybridization with a 1.3-kilobase (kb) sheep elastin cDNA clone [Yoon, K., May, M., Goldstein, N., Indik, Z., Oliver, L., Boyd, C., & Rosenbloom, J. (1984) Biochem. Biophys. Res. Commun. 118, 261-269]. Three clones encompassing 10 kb of the bovine elastin gene were identified and characterized by restriction mapping and DNA sequencing of the 6.2 kb of the most 3' region of the gene. These analyses have permitted localization of eight exons in the 6.2 kb in which the translated exons vary in size from 27 to 69 base pairs, and there is an approximately 1-kb untranslated region at the 3' end. In addition to identification of sequences homologous to those found in porcine tropoelastin, the analyses defined a 58 amino acid sequence that forms the carboxy-terminal region of tropoelastin, and this sequence, which contains two cysteine residues, was previously not observed in the protein sequence data. The analyses also suggest that functionally distinct cross-link and hydrophobic domains of the protein are encoded in separate exons.     

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene.

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML-X and bAML-Y gene with multiple deletions. A pair of sex-specific primers was designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from the X- and Y-chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day-6 to day-7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals.