Molecular cloning of the T4 genome: organization and expression of the tail fiber gene cluster 34--38

Summary T4 derivatives that carry T4 tail fiber genes 34–38 have been isolated and characterized by genetic, structural and functional analysis. 32 T4 recombinants were identified by a marker rescue screen of 310 T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the vectors. The cloned tail fiber genes are expressed efficiently from promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34-gp38 have been detected by SDS polyacrylamide gel electrophoresis of ³⁵S-labeled extracts of appropriate T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34–38 and their mutant variants that affect the assembly, structure and function of the tail fibers.     

Molecular cloning and expression of rat liver bile acid CoA ligase

Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates.     

Molecular cloning and sequencing of human palmitoyl-CoA ligase and its tissue specific expression

A complimentary DNA clone encoding the entire human palmitoyl-CoA ligase has been isolated from a liver cDNA library and sequenced in it's entirety. The predicted product is a 699 amino acid protein. Southern analysis utilizing the human palmitoyl-CoA ligase gene as a probe revealed varying degrees of similarity amongst various mammalian species. The palmitoyl-CoA ligase gene is highly expressed in liver, heart, skeletal muscle and kidney, and to a lesser extent in brain, lung, placenta and pancreas. The expression of palmitoyl-CoA ligase in various tissue parallels the function of this enzyme in the metabolism of fatty acids in these tissues.     

Molecular cloning and expression of the hepatitis delta virus genotype IIb genome

Analysis of hepatitis delta virus (HDV) genome sequences has revealed multiple genotypes with different geographical distributions and associated disease patterns. To date, replication-competent cDNA clones of HDV genotypes I, II, and III have been reported. HDV genotypes I, II, and IIb have been found in Taiwan. Although full-length sequences of genotype IIb have been published, its replication competence in cultured cells has yet to be reported. In order to examine this, we obtained a full-length cDNA clone, Taiwan-IIb-1, from a Taiwanese HDV genotype IIb isolate. Comparison of the complete nucleic acid sequence of Taiwan-IIb-1 with previously published genotype IIb isolates indicated that Taiwan-IIb-1 shares 98% identity with another Taiwanese isolate and 92% identity with a Japanese isolate. Transfection of Taiwan-IIb-1 into COS7 cells resulted in accumulation of the HDV genome and appearance of delta antigens, showing that cloned HDV genotype IIb can replicate in cultured cells.     

Molecular cloning and expression of chondroitin 4-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine residue of chondroitin. The enzyme has been previously purified to apparent homogeneity from the serum-free culture medium of rat chondrosarcoma cells (Yamauchi, A., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J. H., and Habuchi, O. (1999) J. Biol. Chem. 274, 2456-2463). The purified enzyme also catalyzed the sulfation of partially desulfated dermatan sulfate. We have now cloned the cDNA of the mouse C4ST on the basis of the amino acid sequences of peptides obtained from the purified enzyme by protease digestion. This cDNA contains a single open reading frame that predicts a protein composed of 352 amino acid residues. The protein predicts a Type II transmembrane topology. The predicted sequence of the protein contains all of the known amino acid sequence and four potential sites for N-glycosylation, which corresponds to the observation that the purified C4ST is an N-linked glycoprotein. The amino acid sequence of mouse C4ST showed significant sequence homology to HNK-1 sulfotransferase. Comparison of the sequence of mouse C4ST with human HNK-1 sulfotransferase revealed approximately 29% identity and approximately 48% similarity at the amino acid level. When the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that catalyzes the transfer of sulfate to position 4 of GalNAc residue of both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis showed that, among various mouse adult tissues, 5.7-kilobase message of C4ST was mainly expressed in the brain and kidney.     

Molecular cloning and expression of glycoprotein IIIa<Superscript>T1565C</Superscript>

Objective To construct the eukaryotic expression vectors of mutant GPIIIa, establish CHO cell lines stably expressing mutant GPIIIa. Methods Total RNA were extracted from HEL cells. Mutant GPIIIa cDNA was synthesized by RT-PCR using the specific primers designed according to Genbank by Primer 5, then leaded to T1565C. The expression vector pcDNA3.1(+) and PCR products were respectively digested by NheI and HindIII, the specific cDNA fragments were directly inserted to the pcDNA3.1(+) because of having the same adhesive ends. Then wild type pcDNA3.1(+)IIIa and mutant pcDNA3.1(+)IIIa were respectively transfected into CHO cells using Lipofectamine 2000 reagent. The cell lines expressing GPIIIa and GPIIIa^T1565C were screened by G418. Expression of GPIIIa and GPIIIa^T1565C on transfected CHO cell surface were evaluated by flow cytometry and by RT-PCR to substantiate mRNA. Results The cDNAs of GPIIIa and GPIIIa^T1565C were amplified by RT-PCR, and the recombinant of mutant pcDNA3.1(+)IIIa were constructed. By sequencing and enzyme digestion, it was be confirmed that there is a mutant of GPIIIa on 1565(T → C). The result of flow cytometric analysis showed fluorescence intensity in the CHO cells transfected by recombinant is much higher than that by pcDNA3.1(+)IIIa. Conclusions (1) Succeeded in constructing recombinants pcDNA3.1(+)IIIa^T1565C. (2) Succeeded in getting the cell lines expressing GPIIIa^T1565C Supported by Heilongjiang Science & Technology bereau found GB06C40303.     

Molecular architecture and ligand recognition determinants for T4 RNA ligase

RNA ligase type 1 from bacteriophage T4 (Rnl1) is involved in countering a host defense mechanism by repairing 5'-PO4 and 3'-OH groups in tRNA(Lys). Rnl1 is widely used as a reagent in molecular biology. Although many structures for DNA ligases are available, only fragments of RNA ligases such as Rnl2 are known. We report the first crystal structure of a complete RNA ligase, Rnl1, in complex with adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPcPP). The N-terminal domain is related to the equivalent region of DNA ligases and Rnl2 and binds AMPcPP but with further interactions from the additional N-terminal 70 amino acids in Rnl1 (via Tyr37 and Arg54) and the C-terminal domain (Gly269 and Asp272). The active site contains two metal ions, consistent with the two-magnesium ion catalytic mechanism. The C-terminal domain represents a new all alpha-helical fold and has a charge distribution and architecture for helix-nucleic acid groove interaction compatible with tRNA binding.     

Molecular genome organization in ciliates

The review summarizes modern views on to the structure and differentiation of the nuclear apparatus in ciliates. The genetic system of ciliates (type Ciliophora) includes two types of nuclei: germinal micronucleus (MIC) and somatic macronucleus (MAC). The MAC development is associated with the rearrangement of the MIC genome, which includes chromosome fragmentation and chromatin diminution. The loss of DNA constitutes from 10–15% (Tetrahymena termophila) to 95–98% of the genome in spirotrichs (Stylonychia, Oxytricha, and Euplotes). Analysis of molecular mechanisms underlying nuclear dualism in ciliates promoted radical revision of the concept on the interactions and roles of MAC and MIC. The micronucleus, as an inactive element, is an ideal field for the invasion and further expansion of mobile genetic elements. Chromatin diminution plays the purifying role, restoring the native genome structure. The process of recognition of “genetic garbage” to be eliminated has many features in common with the siRNA-mediated heterochromatization. The presence of this mechanism in very early radiated eukaryotic lineages (Opistokonta and Chromalveolata), indicates that it arose at the earliest stages of the eukaryotic evolution, probably, as a mechanism promoting genome integrity and stability.     

Molecular cloning of fragments of bacteriophage T4 DNA

Summary Non-glucosylated T4 DNA was digested with R. EcoRI and the resulting fragments covalently joined to vectors. The genetic content of each -T4 hybrid was determined by marker-rescue tests. The isolation of many recombinants containing partialdigestion products of T4 DNA provided the overlapping sequences necessary to order fragments within the T4 genome. The present analyses include parts of the early region between genes 42 and 46, and much of the late region between genes 50 and 29. T4 cytosine-DNA digested to completion by R.EcoRI was used to identify the fragments of DNA within the -T4 recombinants. The T4 cytosine-DNA was also sensitive to R.HindIII and R.Xho but not to R.BamH1.     

Molecular cloning of the genome of human spumaretrovirus

DNA of human spumaretrovirus (HSRV) was cloned from both cDNA and from viral DNA into phage lambda and bacterial plasmid vectors. The recombinant plasmids harboring viral DNA were characterized by Southern blot hybridization and restriction mapping. Physical maps were constructed from cDNA and found to be colinear with the restriction maps obtained from viral DNA. The recombinant clones isolated contained viral DNA inserts which range in size from 2.2 kb to 15.4 kb. The recombinant clones allowed to construct a physical map of the complete HSRV provirus of 12.2 kb.