Structure of the BoLA‐DRB3 gene and promoter

The cattle major histocompatibility complex (MHC) class II DR gene product is a heterodimer encoded by the BoLA‐DRA and ‐DRB3 genes. Several groups have isolated cDNA and genomic clones for these genes, but their full genomic organization has not been described. We used a combination of long‐range polymerase chain reaction (PCR), cloning and sequencing to define the organization of the DRB3 gene on existing genomic clones and in genomic DNA. We estimate the size of the coding region to be 11.4 kbp. Sequencing of full‐length PCR clones from two different haplotypes confirmed that they carried complete DRB3 genes and allowed the design of probes and primers to isolate and characterize the DRB3 promoter and 3′ end. Fragments carrying the 5′ end of the DRB3 gene and its promoter were identified on bacterial artificial chromosome (BAC) clones carrying the BoLA‐DR genes. A 10‐kbp promoter fragment was subcloned from one clone and a 1.7‐kbp region including exon 1 and the promoter was sequenced. A 3‐kbp fragment encoding exons 4–6 and the entire 3′ untranslated region of the DRB3 gene was isolated from lambda clone A1 and sequenced. This provides us with improved characterization of the DRB3*0101 and DRB3*2002 alleles, and also subcloned 5′ and 3′ flanking regions of the polymorphic DRB3 gene for use in functional studies.     

5' flanking sequence of the human immediate early responsive gene ccn1 (cyr61) and mapping of polymorphic CA repeat sequence motifs in the human ccn1 (cyr61) locus.

AIMS: The human ccn1 (hccn; hcyr61) gene has been identified previously at the mRNA and protein level as a 1,25-dihydroxyvitamin D(3) and growth factor regulated gene in human osteoblasts. This study aimed to analyse genomic clones containing the human ccn1 (cyr61) gene and to provide the 5' flanking region. METHODS: Genomic clones were isolated by screening a lambda library and by array filter hybridisations of a genomic library. Sequencing was performed using the dye terminator method. Promoter activity was measured after transient transfection using a beta galactosidase assay. CA repeat motifs were studied by a combined PCR/fragment analysis protocol. RESULTS: The human 5' flanking region of 870 nucleotides contains several stretches with high homology to the mouse promoter as well as CA repeat motifs. This first report on the human 5' flanking sequence of the hccn1 (hcyr61) gene provides important insights into regulation pathways for the expression of this 1,25-dihydroxyvitamin D(3) and growth factor responsive early gene. A genomic clone containing the hccn1 (hcyr61) gene region also yielded a CA sequence located 3' of the ccn1 (cyr61) gene. This CA repeat and one of the CA repeat motifs in the promoter were studied in detail and found to be polymorphic. CONCLUSIONS: The 5' flanking sequence of the hccn1 (hcyr61) gene provides insights into the mechanisms of regulation of this immediate early gene product. The CA repeat polymorphisms within the gene region will be useful in the genetic study of disorders affecting bone metabolism.     

Structure and chromosomal localization of the human 2',3'-cyclic nucleotide 3'-phosphodiesterase gene

Human brain cDNA clones for the myelin associated enzyme 2' 3' -cyclic nucleotide 3' -phosphodiesterase (CNPase) have been isolated and sequenced. The only 5' untranslated region (UTR) sequence found was that of a human CNPII mRNA, with no direct evidence for a CNPI mRNA. Human CNPase cDNAs were used to isolate genomic clones containing the human CNPase gene which is 9 kb long. Four exons were identified, separated by three introns, and the sequence of each exon and intron/exon boundary has been established.
The polymerase chain reaction (PCR) was used to detect the presence of the human CNPase gene in DNA from a panel of rodent/human somatic cell hybrids. By this means the human CNPase gene was mapped to chromosome 17. In situ hybridization of a human CNPase genomic clone to metaphase chromosomes further localized this gene to chromosomal band 17q21.     

Identification of a Salmonella typhimurium genomic region involved in invasion of HeLa and Henle-407 epithelial cells

To identify the invasion determinant, a cosmid library was constructed by cloning a genomic library of Salmonella typhimurium 82/6915 into a cosmid vector, pLA2917. A genomic region involved in invasion of cultured HeLa and Henle-407 cells was subcloned into plasmid pGEM-7Z. E. coli strain DH1 carrying pSV6235 consisting of a S. typhimurium 4.6 kb genomic region in pGEM-7Z showed invasion of cultured HeLa and Henle-407 cells. Nested sequential deletions were introduced into the 4.6 kb genomic region of pSV6235. The E. coli recombinants which contained less than 1.5 kb deletions from the 5' end (SmaI site) of the genomic region invaded the cells as effectively as DH1 (pSV6235). The invasion of the recombinants carrying over 2.0 kb deletions from the end of pSV6235 was significantly inactivated compared to DH1 (pSV6235). Restriction enzyme analysis showed that the 3.1 kb fragment from the 3' end of the 4.6 kb genomic region was distinguished from the Salmonella pathogenicity I genes of S. typhimurium such as the inv, spa, and hil regions showing invasion of the cultured eukaryotic cells.     

Identification of the recombination site within the steroid 21-hydroxylase gene (CYP21) of the HLA-B47,DR7 haplotype.

The HLA haplotype A3-Cw6-B47-C4A91-BQ0-DR7 is associated with congenital adrenal hyperplasia (CAH), since it only carries the dysfunctional steroid 21-hydroxylase A pseudogene as well as the 5' adjacent complement C4A gene. The recombination site leading to the deletion of the complement C4B and steroid 21-hydroxylase B genes in this haplotype was studied by determining the 21-hydroxylase genomic DNA sequence in comparison to the standard CYP21A- and CYP21B-specific sequences. A 200-bp region between exons 7 and 8 was identified as a possible recombination site. Thus the deleted area comprises the 3' end of the CYP21A pseudogene, the entire C4B gene and the 5' end of the CYP21B gene. The findings were confirmed by PCR amplification of a 1.8-kb fragment of the CYP21 gene. This PCR system is specific for CYP21A/B recombinant genes and may be used for screening among CAH patients carrying this type of deletion.     

Specific amplification of the HLA-DRB4 gene from c-DNA. Complete coding sequence of the HLA alleles DRB4*0103101 and DRB4*01033

We present the complete coding sequence of the HLA alleles DRB4*0103101 and DRB4*01033 derived from the lymphoblastoid cell line G081, established from an individual of Spanish Gypsy ethnic origin. This cell was typed by PCR-SSP and reverse SSO as DRB4*0103101 but further characterization of the DRB4 gene by sequence-based typing (SBT) demonstrated heterozygosity at codon 78 (TAC, TAT). With the aim of confirming this polymorphism, RNA isolated from G081 was subjected to RT-PCR using primers designed to recognize specifically the 5' and 3' UT regions of HLA-DRB4 and the product was cloned and sequenced. Nucleotide sequences derived from seven clones confirmed the heterozygosity of G081, as they corresponded to two open reading frames of 801 nucleotides that matched either DRB4*0103101 or the recently described DRB4*01033, for which a partial sequence, spanning exons 2 and 3, has been reported. The phenotype of G081 (A*01; B*0702, *1302/1303; Cw*0602, *07; DRB1*0403, *0701; DRB4*0103101, *01033; DQB1*0202, *0302; DQA1*0201, *0301) is consistent with a proposed association of DRB4*01033 with DRB1*0403 and DQB1*0302.     

Sequence analysis of the DRB1 promoter reveals limited polymorphism with no influence on gene expression

HLA-class II promoters contain a set of conserved regulatory regions necessary for constitutive and induced gene expression. For the HLA-DQB as well as for the DRB1 promoter sequence, polymorphisms with influence on gene expression have been reported. In contrast to these data we could show that there is very limited allele-specific polymorphism among the HLA-DRB1 promoter alleles. In a long range PCR we amplified a DNA sequence containing the promoter and the second exon of the DRB1 gene in one fragment. Nested PCR products of this PCR fragment for the promoter and for the second exon were analysed by DNA sequencing to allow the linkage of a promoter to its DR allele. Most investigated DRB1 alleles exhibited the same promoter consensus sequence except for two point mutations. An A to T transversion (position -70 bp) was closely associated with DRB1*08, whereas a C-deletion (position -30 bp) was most commonly observed together with DRB1*10. Both polymorphisms did not influence promoter activity in luciferase reporter gene assays.     

2a型丙型肝炎病毒5’非编码区的RACE护增及二级结构的分析

目的：获得真全长中国大陆2a型丙型肝炎病毒（HCV）5’非编码区（5’UTR）cDNA，并分析其一级结构和二级结构，为进一步研究其在HCR复制、翻译中的调控机制、开发新的抗HCV药物奠定基础。方法：利用逆转录套式聚合酶链反应（RT－PCR）联合限制性内切酶长度多态性分析（RFLP）初步筛选出1例2a型HCV感染者，采用cDNA末端快速扩增技术（RACE）扩增出一条约800bp的cDNA片段，A－T克隆，用RFLP与PCR鉴定重组子，全自动序列分析仪测定插入子序列，RNAdraw预测二级结构。结果：RACE法获得真全长2a型HCV 5’端序列。5个克隆中，3个克隆含全长5’UTR序列，与HCV－1，HC－C2，HC－J6，HC－J8相比，同源性分别为93．6％－94．4％，92．1％－93．0％，98．8％－99．7％，96．2％－96．5％，与2a型标准株HC－J6相比，21，170，222，247，339位不同，分别为G，A，C，C，T,但这些突变不影响其二级结构。另外2个克隆为5’端缺失突变株，分别缺失54bp和144bp。结论：RACE技术快速、有效、实用，可用效获得病毒基因组的末端序列；依此获得中国大陆2a型HCV的5’UTR cDNA；在感染者血液中存在5’端部分缺失的HCV基因片段。     

The absence of disease-specific polymorphisms within the HLA-B51 gene that is the susceptible locus for Behçet''s disease

Beh?et's disease is known to be associated with HLA-B51 in many different populations. Genetic evidence supports that the susceptible gene for Beh?et's disease is the HLA-B51 allele at the HLA-B locus. This study was aimed to determine the HLA-B51 nucleotide sequence variation in three Beh?et's disease patients and three healthy controls in order to elucidate if any disease specific mutations or polymorphisms may exist in the HLA-B51 gene of patients. Long-range polymerase chain reaction (PCR) was first carried out to give a PCR-amplified product of 9.5 kb which was then used as a template for nested PCR to give a final amplified product of 4.2 kb. This final product containing the 1.3-kb promoter/enhancer region and the entire HLA-B gene except for a 363-bp 3' terminal end segment encoding the 3' untranslated region was subcloned by the BP cloning technique and sequenced. The sequencing results showed that all the patients possessed the HLA-B*51011 allele, and there were no differences in the exonic nucleotide sequences between the three Beh?et's disease patients and the three healthy controls. The HLA-B*51011 intronic and promoter/enhancer nucleotide sequences from the three patients had 22 single nucleotide polymorphisms (SNPs), a single insertion of 6 bp and a single deletion of 2 bp. On the other hand, the three healthy controls had 24 SNPs in their intronic and promoter/enhancer regions. However, none of these polymorphisms in the patients were specific for the disease. Therefore, these results clearly demonstrate that the HLA-B exonic sequence that encodes the HLA-B51 allele is the real pathogenic factor in Beh?et's disease.     

Organization of human FcRII and FcRII-like (beta FcRII) genes: structural homology to HLA class I and class II genes

Genomic EMBL 3 DNA clones representing part of the human Fc gamma receptor II and the beta Fc gamma receptor II genes were characterized. One of them contains the first five exons including the 5' flanking region of the beta FcRII gene. The signal peptide, the extracellular domains, the putative membrane spanning region, and the first amino acids of the cytoplasmic region encoded by these five exons are spread over approximately 11 kb. Another genomic DNA clone comprises four exons encoding the second extracellular domain and the transmembrane and cytoplasmic regions of the FcRII. Alignment of the genomic DNA clones reveals that these FcR genes are identically organized. Comparison of the corresponding regions of these clones shows that not only the exons are strikingly homologous but also the splice junctions and parts of the intervening sequences are conserved. Furthermore, the genomic organization of FcRII and HLA-class I resemble each other.     

Sequence polymorphisms of the Der p 3 house dust mite allergen

Background The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectricfocusing studies with the natural Der p 3 protein have indicated that several isoforms exist. Objective To determine the extent of the sequence variation of the Der p 3 allergen and distinguish at the molecular level, whether the sequence isoforms represent allelic variants or multiple genes of the allergen. Methods Five cDNA clones of Der p 3 have been isolated from a λ gt 10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PCR analysis of Eco RI digested genomic DNA was performed. Results Southern blot analysis of Eco RI digested genomic DNA showed that the DNA encoding Der p 3 was located on a single 3.5 kb fragment and inverse polymerase chain reaction analysis (PCR) of this DNA showed that there was only a single Der p 3 gene on this 3.5 kb fragment. The nucleotide sequence of one of the clones was identical to the original Der p 3 P3WS1 clone and two clones ditfered only in their 3′untranslated sequences. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conservative. Clone 3 had 98.7% identity with Der p 3 P3WS1. One clone for which the full sequence was not available (clone 4) had only 84.4% identity with the original clone and is therefore consistent with an isoallergen. Conclusions These data along with our previous genomic sequence shows that for the most part, the Der p 3 allergen has only minor sequence variations (variants) although the isoallergen indicated by clone 4 needs further investigation. It is now evident that Der p 3 is encoded by a single gene and that most cDNA clones constructed from commercial mites show only minor sequence variation similar to that observed for the group I and group 2 house dust mite allergens.     

The genomic organization of the mouse CD94 C-type lectin gene.

The mouse natural killer (NK) gene complex is located on chromosome 6 and contains a number of genes encoding C-type lectin receptors which have been found to regulate NK cell function. Among these are CD94 and four NKG2 genes. Like its human counterpart, the mouse CD94 protein associates with different NKG2 isoforms and recognizes the atypical MHC class I molecule Qa-1b. Here, the genomic organization of the mouse CD94 gene was determined by analysing a BAC clone containing the CD94 gene. The mouse CD94 gene contains six exons separated by five introns. Exons I and II encode the 5' untranslated region (UTR) and the transmembrane domain. Exon III encodes the stalk region and exons IV-VI encode the carbohydrate recognition domain (CRD). Furthermore, we cloned and sequenced the CD94 promoter region, and putative regulatory DNA elements were identified. Further studies on the CD94 promoter region may help to elucidate the restricted expression pattern of CD94 in NK cells and a subpopulation of T cells.     

Molecular analysis of the DLA DR region

The dog is an important model for studying organ transplantation. In order to develop a DNA-based typing system, a genomic analysis of the canine DR region of the MHC was undertaken. Southern analysis suggests the presence of two DRB genes in most dogs and all have one DRA gene. One dog out of 200 examined contains only one DRB gene. The DRA gene was mapped in one lambda phage clone. One DRB gene (DRBB1) was localized to two overlapping phage clones. Another DRB gene (DRBB2) was localized to two overlapping phage clones. Sequence analysis of the two DRB genes suggests that one gene is intact (DRBB1) and one gene is a pseudogene (DRBB2) because it lacks a splice acceptor signal for exon 3 and lacks exons 2 and exon 4. Intron 1 and 2 sequence data from DRBB1 allow amplification of the polymorphic exon 2 which, in turn, can serve as a basis for developing a typing system for DRB.     

Immunoglobulin heavy and light chain gene sequences of a human CD5 positive immunocytoma and sequences of four novel VHIII germline genes.

To analyse the V genes expressed by an IgM lambda CD5-positive immunocytoma heavy and light chain V region genes were cloned and sequenced. The heavy chain is composed of a previously undescribed VHIII gene joined to an unknown D gene and to JH4. The light chain V region is composed of a V lambda II gene rearranged to J lambda 1. In an attempt to clone the germline counterpart of the VHIII gene expressed in the immunocytoma PCR amplifications of genomic DNA were carried out and four previously unknown VHIII genes were identified. As several independent clones for the heavy and light chain V region genes were sequenced the rate of somatic mutation of the V genes was calculated to be below 2 x 10(-5)/bp/cell division.