Structure and evolutionary origin of the human granzyme H gene

Among the molecules proposed to be involved in cytotoxic T lymphocyte (CTL), natural killer (NK) and lymphokine activated killer (LAK) cell-mediated lysis are the granzymes, a family of serine proteases stored in the cytoplasmic granules of CTLs, NK and LAK cells. In addition to the granzymes A and B, a third member of this family has been cloned in man and designated granzyme H. We present the complete gene sequence including the 5' promoter region and demonstrate that the granzyme H sequence represents a functional gene expressed in activated T cells. Granzyme H shows the highest degree (greater than 54%) of amino acid sequence homology with granzyme B and cathepsin G and, like these genes, consists of five exons separated by introns at equivalent positions. The evolutionary history of granzyme H has been analyzed by reconstructing an evolutionary tree for granzyme sequences. We provide evidence that interlocus recombination between the ancestral genes of granzyme B and granzyme H occurred about 21 million years ago, leading to a replacement of exon 3, intron 3 and part of exon 4 in human granzyme H by human granzyme B sequences. Our results suggest that the ancestral gene of granzyme H is more closely related to cathepsin G and granzyme B than to the murine granzymes C to G. Thus, granzyme H does not represent a human counterpart of the known murine granzymes A to G. It diverged from cathepsin G before mammalian radiation and should, therefore, exist in other mammalian lineages as well.     

Rapid, comprehensive screening of the human medium chain acyl-CoA dehydrogenase gene

Newborn screening by tandem mass spectrometry (MS/MS) identifies patients with medium chain acyl-CoA dehydrogenase (MCAD) deficiency the most frequently observed disorder of fatty acid oxidation. Molecular genetic analysis is becoming a common tool to confirm those identified as affected by prospective screening and for carrier detection in family studies. The A985G (K304E) mutation accounts for approximately 80% of mutant alleles in MCAD deficient patients, presenting symptomatically, while greater variability of mutant alleles is observed among cases identified through prospective screening. Aside from A985G, the mutation spectrum in MCAD deficient patients is heterogeneous such that comprehensive gene analysis is required. Traditionally the MCAD gene is assayed by sequencing the entire coding region. Although effective and definitive, this approach is expensive, turn around time is slow, and is poorly amenable to a clinical service molecular genetics laboratory. Dye-binding/high-resolution thermal denaturation is a rapid and homogeneous method by which to scan a PCR product for evidence of sequence aberration. PCR is performed in capillaries in the presence of the dsDNA-binding dye LCGreen I and subsequently the DNA/dye complexes are analyzed by high-resolution thermal denaturation. DNA sequencing was limited to fragments displaying abnormal melting profiles. Of 18 specimens analyzed, 11 have a genotype consistent with MCAD deficiency and seven have a genotype consistent with carrier status. Clinical and biochemical data corroborate that the genotype results identified the affected patients and differentiates them from carriers. The entire process is homogeneous requiring no post-PCR manipulation and is completed in under 3 h.     

Structure and expression of the human pseudoautosomal gene XE7.

The human pseudoautosomal region comprises a 2.6 megabase segment of the distal short arms of the X and Y chromosomes. Complete DNA sequence homology between the two sex chromosomes is found in this region, and is believed to be important in mediating X-Y pairing in male meiosis. The only known functional genes in this region are MIC2 and CSF2R; in addition a pseudoautosomal location has been proposed for a genetic locus controlling stature. Here we report the structure of a recently identified pseudoautosomal gene, XE7, and its expression in human tissues. Analysis of genomic and cDNA clones shows that alternative RNA splicing results in the production of two predicted protein isoforms, one containing 385 amino acids and the other with 695 residues. The smaller polypeptide is a truncated version of the larger, and results from the inclusion of a cassette exon that introduces an in-frame stop codon into the mRNA. The XE7 gene appears to be ubiquitously expressed, and the production of both protein isoforms is predicted in each of several tissues examined.     

A novel missense mutation in human lactate dehydrogenase B-subunit gene

Reduced activity of serum lactate dehydrogenase (LDH; EC 1.1.1.27) was found in a male medical student during practical examinations of his own blood. Serum LDH isoenzyme pattern showed reductions in activities of the isoenzymes with lower subunit A/B ratios such as LDH1 and LDH2. These findings were indicative of a partial LDH-B subunit deficiency, which was confirmed in erythrocyte hemolysates by Western blotting. Polymerase chain reaction (PCR)-based DNA sequence analysis of the LDH-B subunit gene revealed a heterozygous nucleotide change: a guanine to adenine substitution in codon 69 (GGG --> GAG) at the third exon of the LDH-B subunit gene that resulted in a glycine to glutamic acid substitution (G69E). The mutation was confirmed by PCR-restriction fragment length polymorphism (RFLP) analysis using a mismatched primer to introduce a new NcoI restriction site. The same heterozygous mutation was found in his mother but not in other family members. This mutation involves a residue belonging to alphaC helix in LDH-B subunit protein molecule that functions as an interface for other subunits.     

Independent origin of single and double mutations in the human glucose 6-phosphate dehydrogenase gene

The vast majority of both polymorphic and sporadic G6PD variants are due to single missense mutations. In the four polymorphic variants that have two point mutations, one of the mutations is always 376 A→G (126 Asn→Asp), which on its own gives rise to the nondeficient polymorphic variant, G6PD A. In a study of G6PD deficient patients who presented with clinical favism in Spain, we have found a new polymorphic variant that we have called G6PD Malaga, whose only abnormality is a 542 A→T (181 Asp→Val) mutation. This is the same mutation as previously found in association with the mutation of G6PD A in the double mutant, G6PD Santamaria. G6PD Malaga is associated with enzyme deficiency (class III), and the enzymic properties of G6PD Malaga and G6PD Santamaria are quite similar, indicating that in this case the effects of the two mutations are additive rather than synergistic. G6PD Santamaria might have been produced by recombination between G6PD A and G6PD Malaga; however haplotype analysis, including the use of a new silent polymorphism, suggests that the same 542 A→T mutation has taken place independently in a G6PD B gene to give G6PD Malaga and in a G6PD A gene to give G6PD Santamaria. These findings help to outline the relationship and evolution of mutations in the human G6PD locus. © 1996 Wiley-Liss, Inc.     

Cloning and characterization of a novel intronless lactate dehydrogenase gene in human testis

Using cDNA microarray hybridization from a human testicular cDNA library, one gene named lactate dehydrogenase A-like gene (LDHL, also known as LDHL6B) was cloned. LDHL exhibited 3.8-fold difference at expression level between adult and fetal human testes. The full cDNA length of LDHL is 1680 bp and had a 1145 bp open reading frame, which encoded a 41.9 kDa protein of 381 amino acids. Sequence analysis showed that LDHL harbors all the domains (one lactate/malate dehydrogenase, NAD binding domain and one lactate/malate dehydrogenase, alpha/beta C-terminal domain) in lactate dehydrogenase gene family. Blasting human genome database localized LDHL to human chromosome 15q22.2 and it was an intronless gene. Results of multiple-tissue PCR and real-time PCR showed that LDHL expressed mainly in testis and its mRNA abundance was testis development-related. In summary, LDHL is believed to be involved in testis development and spermatogenesis.     

三例谷氨酸脱氢酶型先天性高胰岛素血症患儿的GLUD1基因突变分析

目的 对3例临床诊断为谷氨酸脱氢酶型先天性高胰岛素血症的患儿家系进行遗传学分析并对中国儿童谷氨酸脱氢酶型先天性高胰岛素血症的遗传发病机制进行初步探讨.方法 选取3例临床诊断为谷氨酸脱氢酶型先天性高胰岛素血症患儿的家系,应用PCR-DNA直接测序技术对3例患儿家系谷氨酸脱氢酶1基因(glutamate dehydrogenase 1,GLUD1)第6,7,10,11,12外显子区进行测序分析.结果 第1例患儿及其父亲GLUD1基因第7外显子区发现了1个R269H杂合突变,遗传方式为常染色体显性遗传.第2例患者GLUD1基因的第11外显子区发现了1个S445L杂合突变,为新生突变.第3例患儿家系GLUD1基因第6,7,10,11,12外显子均未发现突变.结论 在某些中国人中,谷氨酸脱氢酶基因R269H,S445L杂合突变可以导致谷氨酸脱氢酶型先天性高胰岛素血症的发生.     

Structure and function of ASP, the human homolog of the mouse agouti gene

The mouse agouti coat color gene encodes a novel paracrine signalingmolecule whose pulsatile expression produces a characteristicpattern of banded pigment in individual hairs. Several spontaneousagouti alleles produce adult-onset obesity and diabetes, andhave provided important single-gene animal models for alterationsin energy metabolism. Utilizing linkage groups conserved betweenmice and humans, we have cloned the human homolog of the mouseagouti gene from a human chromosome 20 yeast artificial chromosomeknown to contain S-adenosyl homocysteine hydrolase (AHCY). Thehuman agouti gene, named Agouti Signaling Protein (ASP), encodesa 132 amino acid protein, the mRNA for which is expressed intestis, ovary, and heart, and at lower levels in liver, kidney,and foreskin. As predicted by the interactions of mouse agoutiwith the extension gene (which encodes the melanocyte receptorfor alpha-melanocyte stimulating hormone [     

Regional localization of the sperm-specific lactate dehydrogenase, LDHC, gene on human chromosome 11

A cDNA clone complementary to the mRNA encoding the sperm-specific lactate dehydrogenase, LDHC, has been used to map the LDHC locus to the short arm of human chromosome 11. In situ hybridization data and analysis of mouse/human somatic cell hybrids carrying deletions of human chromosome 11 suggest that the gene is localized at p15.3-p15.5 close to the LDHA gene.     

人乙醛脱氢酶2基因在毕赤酵母SMD1168中的表达研究

目的得到一株产人乙醛脱氢酶2(acetaldehyde dehydrogenase-2,ALDH2)的重组毕赤酵母菌株,优化其发酵条件以满足生产和科研需求。方法将ALDH2基因整合到质粒pPIC9K,构建重组表达载体pPIC9K-ALDH2,将重组表达质粒pPIC9K-ALDH2经电转化到毕赤酵母SMD1168中,转化子经MD平板筛选后,用G418筛选多拷贝重组子,测序鉴定。通过改变诱导温度、pH、初始OD值、甲醇浓度等诱导条件,对SMD1168(pPIC9K-ALDH2)发酵的条件进行优化。结果ALDH2cDNA整合到毕赤酵母基因组中,得到SMD1168(pPIC9K-ALDH2);获得最佳的诱导表达优化条件:在28℃、pH=7.0、诱导初始OD600=12、每隔24h添加1.5%甲醇、转速为300r/min,发酵所产生的人ALDH2酶活可达到0.115U/ml,相对较高。结论以蛋白酶缺陷型毕赤酵母SMD1168为宿主,分泌表达质粒pPIC9K为载体,能够成功构建重组子SMD1168(pPIC9K-ALDH2)。     

Cloning and transcription analysis of the Candida rugosa propionyl-CoA dehydrogenase gene and its expression in Pichia pastoris

The propionyl-CoA dehydrogenase (PACD) gene was cloned from Candida rugosa by the cDNA RACE technique. The full cDNA of the PACD gene has a length of 1408 bp, which contains a complete open reading frame (ORF) of 1329 bp, coding for 442 amino acids. The cDNA of PACD was cloned into the expression plasmid pPIC9K and transformed into Pichia pastoris GS115. The recombinant protein was purified by Ni-NTA affinity chromatography, and its size was observed to be approximately 49 kDa as estimated by SDS-PAGE. Anti-His antibodies were used to characterise the recombinant PACD by western-blot analysis. The recombinant protein retained the activity of catalysing propionyl-CoA to acryloyl-CoA. The results of dot-blotting hybridisation using a PACD cDNA probe indicated that the PACD mRNA level was modified at different stages: mRNA levels were low for the first 36 h, then increased through 48 h and eventually reached a stable level. These results indicate that propionate induction could significantly activate PACD mRNA expression. Information from this study will be helpful in elucidating the metabolic pathway for 3-hydroxypropionic acid production in C. rugosa.     

Mutational analysis of the ARAF gene in human cancers

Deregulation of RAS signal transduction has been implicated in the malignant growth of human cancer cells. The BRAF gene, encoding a RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers, raising the possibility that other RAF family members might be mutated in human cancers. In this study we analyzed the genomic DNAs for the detection of somatic mutations of the ARAF gene in 60 human cancer cell lines and 323 primary human cancer tissues, including colorectal carcinomas, gastric carcinomas, ovarian tumors and acute leukemias. The MOLT-4 leukemia cell line was found to harbor an ARAF gene mutation resulting in an amino acid substitution (A451T) at the activation segment in the kinase domain of ARAF. In the cancer tissues we could not detect any ARAF gene mutation. Our data indicate that, in contrast to the BRAF gene, the ARAF gene is rarely mutated in human cancers, and suggest that alterations of the RAS pathway by ARAF gene mutation may not play an important role in the pathogenesis of human cancers.     

An analysis of the human and mouse CXCR5 gene introns

Both mouse and human chemokine receptor CXC motif 5 (CXCR5) genes exhibit one single intron interrupting the coding sequence. The mouse intron is 12053 nucleotides (nt) long; the human intron is 9603 nt long. Sections of the mouse intron significantly align plus/plus with sections of the human intron; the aligned segments are in the same order in mouse as in man and overall cover 13% of the mouse sequence and 17% of the human sequence. The human CXCR5 intron harbors sequences derived from retroviruses (human endogenous retroviruses). The mouse intron comprises very similar sequences. About 70% of the mouse intron sequence is ‘specific’ to this gene, while sequences in the rest of the intron are shared with many other genes located on different chromosomes. In the human the coverage by specific sequences is about 87%. Thus, the contribution of transposable elements is significantly higher in mouse (30%) than in man (13%). Intra-intronic plus/minus alignments exist in mouse (10 couples) and man (two couples): these may form stem and loop structures determining the secondary structure of the corresponding pre-mRNAs.