The cloning,genomic organization and tissue expression profile of the human DLG5 gene

Background  

Familial atrial fibrillation, an autosomal dominant disease, was previously mapped to chromosome 10q22. One of the genes mapped to the 10q22 region is DLG5, a member of the MAGUKs (Membrane Associated Gyanylate Kinase) family which mediates intracellular signaling. Only a partial cDNA was available for DLG5. To exclude potential disease inducing mutations, it was necessary to obtain a complete cDNA and genomic sequence of the gene.     

pH-rate profiles of l-arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in l-xylulose production

l-Arabinitol 4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of l-arabinitol oxidation by NAD⁺, catalyzed by HjLAD, was studied within the pH range of 7.0–9.5 at 25 °C. The turnover number (k_cat) and the catalytic efficiency (k_cat/K_m) were 4200 min⁻¹ and 290 mM⁻¹ min⁻¹, respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare l-sugar l-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%.     

The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution

The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 Å resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted β-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61.     

The bovine interleukin-4 gene: genomic organization,localization, and evolution

Interleukin-4 (IL4) is involved in the immune response to certain parasites and possibly in the development of some atopic diseases since it triggers the T helper 2 lymphocyte response. Therefore, IL4 is a candidate gene, for example, for disease association studies and gene mapping. We isolated bovine IL4 cosmids and determined the genomic organization. Fragments carrying the exons as well as 725 base pairs (bp) from the 5 flanking and 190 bp from the 3 flanking region were cloned and sequenced. The first 481 base pairs of the 5 flanking region, including the putative promoter sequences, are surprisingly similar (92%) between cattle and human. In addition, we cloned and sequenced a mixed [(t/g)a]_m(ca)_n repeat located approximately 35 kilobases upstream from the IL4 gene. It showed seven repeat length alleles in a limited number of animals. The IL4 gene has been assigned to 7q15-q21 by fluorescence in situ hybridization in cattle. Evolutionary aspects are discussed on the basis of sequence data as well as interspecies chromosomal homologies.     

The mouse Kell blood group gene (Kel): cDNA sequence, genomic organization, expression, and enzymatic function

The human Kell blood group system is important in transfusion medicine, since Kell is a polymorphic protein and some of its antigens can cause severe reactions if mismatched blood is transfused, while maternal alloimmunization may lead to fetal and neonatal anemia. In humans, Kell is an Mr 93,000 type II membrane glycoprotein with endothelin-3-converting enzyme activity that is linked by a single disulfide bond to another protein, XK, that spans the membrane ten times. An absence of XK leads to clinical symptoms termed the McLeod syndrome. We determined the cDNA sequence of the mouse Kell homologue, the organization of the gene, expression of the protein and its enzymatic function on red cells. Comparison of human and mouse Kell cDNA showed 80% nucleotide and 74% amino acid sequence identity. Notable differences are that the mouse Kell protein has eight probable N-linked carbohydrate side chains, compared to five for human Kell, and that the mouse homologue has one more extracellular cysteine than human Kell protein. The mouse Kell gene (Kel), like its human counterpart, is similarly organized into 19 exons. Kel was located to proximal Chromosome 6. Northern blot analysis showed high expression in spleen and weaker levels in testis and heart. Western blot analysis of red cell membrane proteins demonstrated that mouse Kell glycoprotein has an apparent Mr of 110,000 and, on removal of N-linked sugars, 80,000. As in human red cells, Kell is disulfide-linked to XK and mouse red cells have endothelin-3-converting enzyme activity.     

Complete cDNA sequence,expression, alternative splicing,and genomic organization of the mouse Nfat5 gene

Members of the NFAT (nuclear factors of activated T cells) gene family have been investigated in numerous organisms, including man and mouse. All NFATs may be synthesized in several isoforms differing in amino or carboxy termini due to 5' and 3' alternative splicing of the corresponding mRNA. Recently, we mapped the murine Nfat5 gene to chromosome 8D. In the present paper we describe for the first time the complete sequence and primary structure of murine Nfat5, two new spliced isoforms, and the expression of murine Nfat5 in embryonic and adult mouse tissues.     

Cloning and expression of 5, 10-Methylenetetrahydrofolate reductase (MTHFR) gene

Hyperhomocysteinemia is an independent risk factor of cardiovascular diseases and birth defects. One of the important factors causing hyperhomocysteinemia is decrease of 5,10-Methylenetetrahydrofdate reductase. Human and rat MTHFR cDNAs with RT-PCR were isolated, a prokaryodytic expression vector containing human MTHFR cDNA was constructed, and human MTHFR protein was expressed inE. coli. It was also found that the expression of rat MTHFR could be promoted by IL-1 and homocysteine.     

In vivo expression and genomic organization of the mouse cyclin I gene (Ccni)

Cyclins control cell-cycle progression by regulating the activity of cyclin-dependent kinases. Cyclin I was recently added to the cyclin family of proteins because of the presence of a cyclin box motif in the deduced amino-acid sequence. Cyclin I may share functional roles with cyclin G1 and G2 because of the high structural similarity between their deduced amino-acid sequences. However, the biological and functional roles of this subclass of cyclins remain obscure. The mouse cyclin G1 and G2 genes have previously been cloned and characterized. In this report, we describe the cloning of the mouse homolog of cyclin I. The cyclin I cDNA sequence was used to determine the genomic organization of the mouse cyclin I gene which co-localizes with cyclin G2 to chromosome 5E3.3-F1.3. Cyclin I was transcribed from seven exons distributed over more than 19kb of genomic sequence. The expression of cyclin I was determined in various tissues, but no clear correlation with the proliferative state was found. Furthermore, in contrast to cyclin G1, cyclin I expression was stable during cell-cycle progression after partial hepatectomy in both the absence and presence of DNA damage. Transient expression of cyclin I-green fluorescent protein (GFP) fusion proteins in cell lines showed that cyclin I was distributed throughout the cell in contrast with the mainly cytoplasmic localization of cyclin G2 and nuclear localization of cyclin G1. Our results indicate that despite the close structural similarity between cyclin G1, G2 and I, these three proteins are likely to have distinct biological roles.     

酵母菌色氨酸合成酶基因的克隆与表达

用RemHI酶切酿酒酵母(Saceharomyces cercuisiae) 1412-4D染色体DNA,通过蔗糖梯度分离2-4kb DNA片段并插入穿棱质粒pCN60,构成1412-4D基因文库。从基因文库中提取重组质粒,转化受体菌C9(a,trp5,adcl,ade6),用直接功能互补法,分离到9株重组质粒,它们都含有3．2kb的TRP5 DNA片段,分别命名为pCN60(trps)1-90转化体中色氨酸合成酶的酶活水平比原始菌株1412-4D高3倍。