Identification of carriers of the mutation causing coagulation factor XI deficiency in Polish Holstein-Friesian cattle

Factor XI (FXI) deficiency is a hereditary coagulation disorder observed in various mammalian species. The molecular basis of coagulopathy has been recognized in Holstein cattle as a 76-bp insertion in the coding region of theFXI gene. Because the disorder seems to have an impact on reproductive traits and udder health in cattle, we tested 103 randomly selected cows, 28 cows with repeat breeding, and 9 cows with recurrent mastitis for the presence of an abnormalFXI allele. Three related cows were diagnosed as carriers.     

Cloning of cDNAs encoding human interphotoreceptor retinoid-binding protein (IRBP) and comparison with bovine IRBP sequences

We have determined the sequence of the human interphotoreceptor retinoid-binding protein mRNA from three separately isolated cDNAs. The sequence is 4.28 kb long and encodes a protein of 1247 amino acids (aa) including a putative signal peptide and propeptide. The sequence is shorter (by about 1.67 kb) than the bovine mRNA with the major difference in the lengths located in the 3'-untranslated region. We suggest that this resulted from an insertion in the bovine gene or a large deletion from the human gene. The insertion/deletion is flanked on either side by sequences that are similar in the bovine and human sequences. Like the bovine polypeptide, the deduced protein sequence from the human cDNA contains a fourfold repeat, with each repeat containing about 300 aa. Among the four repeats, the identity is about 30-40%. The identity between the complete bovine and human polypeptide sequences is 84%. The identity between the nucleotide sequences is 83% (excluding the major insertion/deletion). Comparison with the bovine gene indicates that the human sequence may lack about 5-10 bp at the 5' end of the cDNA; it, however, includes a poly(A) tail at the 3' end. Thus, the human sequence is virtually full length, is similar to the bovine sequence, and contains a striking fourfold repeat.     

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.     

Sequence variation in the bovine and ovine PRNP genes

A resequencing approach was adopted to identify sequence variants in the PRNP gene that may affect susceptibility or resistance to bovine spongiform encephalopathy. The entire PRNP gene (>21 kb) was sequenced from 26 chromosomes from a group of Holstein-Friesian cows, as well as exon 3 of PRNP (>4 kb) from a further 24 chromosomes from six diverse breeds. We identified 51 variant sequences of which 42 were single nucleotide polymorphisms and nine were insertion/deletion (indel) events. The study was extended to exon 3 of the sheep PRNP gene where 23 sequence variants were observed, four of which were indels. The level of nucleotide diversity in the complete bovine PRNP gene was pi = 0.00079, which is similar to that found at the bovine T-cell receptor alpha delta joining region (pi = 0.00077), but somewhat less than that observed for the bovine leptin (pi = 0.00265). Sequence variation within exon 3 of PRNP in both cattle (pi = 0.00102) and sheep (pi = 0.00171) was greater than that for the complete PRNP gene, with sheep showing greater sequence variation in exon 3 than cattle. The level of sequence variation reported here is greater than previously thought for the bovine PRNP gene in cattle. This study highlights the contribution that recombination plays in increasing allelic diversity in this species.     

Gene structure of human and mouse methylenetetrahydrofolate reductase (MTHFR)

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. A human cDNA for MTHFR, 2.2 kb in length, has been expressed and shown to result in a catalytically active enzyme of approximately 70 kDa. Fifteen mutations have been identified in the MTHFR gene: 14 rare mutations associated with severe enzymatic deficiency and 1 common variant associated with a milder deficiency. The common polymorphism has been implicated in three multifactorial diseases: occlusive vascular disease, neural tube defects, and colon cancer. The human gene has been mapped to chromosomal region 1p36.3 while the mouse gene has been localized to distal Chromosome (Chr) 4. Here we report the isolation and characterization of the human and mouse genes for MTHFR. A human genomic clone (17 kb) was found to contain the entire cDNA sequence of 2.2 kb; there were 11 exons ranging in size from 102 bp to 432 bp. Intron sizes ranged from 250 bp to 1.5 kb with one exception of 4.2 kb. The mouse genomic clones (19 kb) start 7 kb 5′ exon 1 and extend to the end of the coding sequence. The mouse amino acid sequence is approximately 90% identical to the corresponding human sequence. The exon sizes, locations of intronic boundaries, and intron sizes are also quite similar between the two species. The availability of human genomic clones has been useful in designing primers for exon amplification and mutation detection. The mouse genomic clones will be helpful in designing constructs for gene targeting and generation of mouse models for MTHFR deficiency. Received: 28 January 1998 / Accepted: 9 April 1998     

CYP21/C4 gene organisation in Italian 21-hydroxylase deficiency families

Summary A total of 33 Italian 21-hydroxylase (21-OH) deficiency families were investigated using a combination of short and long range restriction mapping of the CYP21/C4 gene cluster. The analyses revealed that large-scale length polymorphism in this gene cluster strictly conformed to a compound variable number of tandem repeats (VNTR) plus insertion system with between one and four CYP21 + C4 units and seven BssHII restriction fragment length polymorphisms (RFLPs) (75kb, 80kb, 105kb, 110kb, 135kb, 140kb and 180kb). A total of 9/66 disease haplotypes, but only 1/61 nondisease haplotypes, showed evidence of gene addition by exhibiting three or more CYP21 + C4 repeat units. Of these, two were identified in one 21-OH deficiency patient who has a total of eight CYP21 + C4 units, being homozygous for the HLA haplotype DR2 DQ2 B5 A28. This haplotype carries four CYP21 + C4 units, three of which contain CYP21A-like genes and one of which contains a CYP21B-like gene that presumably carries a pathological point mutation. Of the other gene addition haplotypes associated with 21-OH deficiency, four show three CYP21 + C4 units flanked by HLA-DR1 and HLA-B14 markers. Although such haplotypes have commonly been associated with non-classical 21-OH deficiency, three examples in the present study are unexpectedly found in two salt-wasting patients, who are respectively homozygous or heterozygous for this haplotype. Only 7/66 disease haplotypes showed evidence of a CYP21B gene deletion.     

Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus

Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.     

Interphotoreceptor retinoid-binding protein. Gene characterization, protein repeat structure, and its evolution

The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene.     

中国荷斯坦牛白细胞黏附缺陷症PCR-RFLP检测方法的研究

本试验根据已知牛染色体上CD18编码基因序列设计引物,提取牛血液和精液DNA,可扩增出338bp的DNA片段,将PCR产物克隆到pMD18-T载体中,对阳性重组质粒进行测序,确定为牛的CD18基因。由于CD18基因的383位碱基由A变为G,而引起牛白细胞黏附缺陷症(BLAD),通过对济南市11个奶牛场356头奶牛及53头荷斯坦种公牛进行了BLAD的PCR-RFLP检测,共检出3头杂合母牛(携带者),占检测母牛群的0.84%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者。     

The human apolipoprotein A-II gene: complete nucleic acid sequence and genomic organization.

The gene for human apolipoprotein (apo) A-II has been isolated from a human genomic DNA library. The cloned fragment was approximately 14 kilobase-pair (kb) long, and extended about 9.0 kb upstream as well as 3.5 kb downstream from the apoA-II gene, which was contained within a 3.1 kb HindIII fragment of human DNA. The complete nucleic acid sequence of the apoA-II gene has been determined, establishing that the apoA-II gene is interrupted by three intervening sequences of 182, 293, and 395 bp. The second intron is of particular interest, because it contains a 33 bp sequence of alternating G and T residues very close to the 3' splice site which has the potential to form a left handed Z-helix structure in vivo. A restriction fragment length polymorphism 3' from the apoA-II gene has been detected which may serve as a marker for the long arm of chromosome 1 in linkage analyses.     

Analysis and frequency of bovine lymphocyte antigen (BoLA-DRB3) alleles in Iranian Holstein cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11 and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2 and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4% and 4.4%, respectively. Thus in the Iranian Holstein cows studied are found alleles which are associated with resistance to various diseases. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.     

Sequence arrangement of the rDNA of Drosophila melanogaster.

The sequence arrangement of genes coding for stable rRNA species and of the interspersed spacers on long single strands of rDNA purified from total chromosomal DNA of Drosophila melanogaster has been determined by a study of the structure of rRNA:DNA hybrids which were mounted for electron microscope observation by the gene 32-ethidium bromide technique. One repeat unit contains the following sequences in the order given. First, an 18 S gene of length 2.13 +/- 0.17 kb. Second, an internal transcribed spacer (Spl) of length 1.58 +/- 0.15 kb. A short sequence coding for the 5.8S and perhaps the 2S rRNA species is located within this spacer. Third, the 28S gene with a length of 4.36 +/- 0.23 kb. About 55% of the 28S genes are unbroken or continuous (C genes). However, about 45% of the 28S genes contain an insertion of an additional segment of DNA that is not complementary to rRNA (l genes). The insertion occurs at a reproducible point 2.99 +/- 0.26 kb from the junction with Spl. The insertions are heterogeneous in length and occur in three broad size classes: 1.42 +/- 0.47, 3.97 +/- 0.55, and 6.59 +/- 0.62 kb. Fourth, an external spacer between the 28S gene and the next 18S gene which is presumably mainly nontranscribed and which has a heterogeneous length distribution with a mean length and standard deviation of 5.67 +/- 1.92 kb. Short inverted repeat stems (100-400 nucleotide pairs) occur at the base of the insertion. It is known from other studies that I genes occur only on the X chromosome. The present study shows that the I and C genes on the X chromosomes are approximately randomly assorted. The sequence arrangement on the plasmid pDm103 containing one repeat of rDNA (Glover et al., 1975) has been determined by similar methods. The I gene on this plasmid contains an inverted repeat stem. The occurrence of inverted repeat sequences flanking the insertion supports the speculation that these sequences are translocatable elements similar to procaryotic translocons.     

Comparative analysis of the bovine MHC class IIb sequence identifies inversion breakpoints and three unexpected genes

The bovine major histocompatibility complex (MHC) or BoLA is organized differently from typical mammalian MHCs in that a large portion of the class II region, called class IIb, has been transposed to a position near the centromere on bovine chromosome 23. Gene mapping indicated that the rearrangement resulted from a single inversion, but the boundaries and gene content of the inverted segment have not been fully determined. Here, we report the genomic sequence of BoLA IIb. Comparative sequence analysis with the human MHC revealed that the proximal inversion breakpoint occurred approximately 2.5 kb from the 3' end of the glutamate-cysteine ligase, catalytic subunit (GCLC) locus and that the distal breakpoint occurred about 2 kb from the 5' end from a divergent class IIDRbeta-like sequence designated DSB. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared with the corresponding region of the human class II MHC (HLA class II). Differences with HLA include the presence of a single histone H2B gene located between the proteasome subunit, beta type, 9 (PSMB9) and DMB loci and a duplicated TAP2 with a variant splice site. BoLA IIb spans approximately 450 kb DNA, with 20 apparently intact genes and no obvious pseudogenes. The region contains 227 simple sequence repeats (SSRs) and approximately 167 kb of retroviral-related repetitive DNA. Nineteen of the 20 genes identified in silico are supported by bovine EST data indicating that the functional gene content of BoLA IIb has not been diminished because it has been transposed from the remainder of BoLA genes.     

The 3' untranslated region of picornavirus RNA: features required for efficient genome replication.

The role of the 3' untranslated region (3'UTR) in the replication of enteroviruses has been studied with a series of mutants derived from either poliovirus type 3 (PV3) or a PV3 replicon containing the reporter gene chloramphenicol acetyltransferase. Replication was observed when the PV3 3'UTR was replaced with that of either coxsackie B4 virus, human rhinovirus 14 (HRV14), bovine enterovirus, or hepatitis A virus, despite the lack of sequence and secondary structure homology of the 3'UTRs of these viruses. The levels of replication observed for recombinants containing the 3'UTRs of hepatitis A virus and bovine enterovirus were lower than those for PV3 and the other recombinants. Extensive site-directed mutagenesis of the single stem-loop structure formed by the HRV14 3'UTR indicated the importance of (i) the loop sequence, (ii) the stability of the stem, and (iii) the location of the stem immediately upstream of the poly(A) tail. The role of a 4-bp motif at the base of the HRV14 stem, highly conserved among rhinoviruses, was examined by site-directed mutagenesis of individual base pairs. This analysis did not pinpoint a particular base pair as crucial for function. The requirement for immediate adjacent positioning of the open reading frame and the 3'UTR was examined by insertion of a 1.1-kb heterologous sequence. A replicon containing this insert replicated to about 30% of the level observed for the wild type. However, the corresponding virus consistently deleted most of the inserted fragment, suggesting that its presence was incompatible with a full replication cycle.