Gene 26 of bacteriophage T4 basal plate. I. Two expression products of the cloned gene

We have cloned the 1.9 kb EcoRV-BglII DNA fragment with T4 genes 51, 26, and 25 into the expression plasmid pT7-5 carrying a T7 promoter. The resulting recombinant plasmid, pRR5-3, contained T4 genes 26 and 25 in the correct orientation for expression. We expressed these genes using the T7 RNA polymerase/promoter system and the synthesis of three polypeptides with the molecular masses of approximately 24, 15, and 8-9 kDa was observed. Expression of genes from the subcloned DNA fragments and from the fragments carrying deletions was studied as well and the 15 kDa protein appeared to be the product of gene 25, while 24 kDa and 8-9 kDa proteins were identified as products of gene 26. The 8-9 kDa protein was shown to be expressed from the end region of gene 26. Having analysed the proteins expressed from the fragments carrying fusion of genes 26 and 25 we supposed two products of gene 26 to be encoded by the same open reading frame.     

Location and identification of the genes for adenovirus type 2 early polypeptides

Virus-specific RNA was prepared from cells early after adenovirus type 2 infection and fractionated by hybridization to specific fragments of viral DNA. The viral mRNA was used to program cell-free protein synthesis, and the products were analyzed by electrophoresis. The genes for the early polypeptides of apparent molecular weight 44,000, 15,000, 72,000, 15,500, 19,000, and 11,000 daltons were located, respectively, between positions 0–4.1, 4.1–16.7, 58.5–70.7, 75.9–83.4, 89.7–98.6, and 89.7–98.6 of the conventional adenovirus DNA map. The polypeptide of molecular weight 72,000 daltons was shown to be the single-strand DNA-binding protein described by others. RNAs from three different adeno-transformed cell lines each program the synthesis in vitro of predominantly the 15K polypeptide, as well as variable amounts of the polypeptide of molecular weight 44,000 daltons. The genes for these two polypeptides are located in the portion of DNA known to be required for transformation of rodent cells by adenovirus.     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

Mapping of the structural gene of pseudorabies virus glycoprotein A and identification of two non-glycosylated precursor polypeptides.

Cell-free translation of pseudorabies virus RNA isolated during the late phase of the infectious cycle yielded a variety of polypeptides. A monoclonal antibody directed against one of the major viral glycoproteins, gA, immunoprecipitated two polypeptides ranging in molecular weight from 78K to 83K. To localize the structural gene for gA, we used cloned BamHI fragments of the viral DNA to select specific mRNA species and immunoprecipitated their in vitro translation products with the anti-gA monoclonal antibody. This allowed us to map the genomic region encoding the mRNA for the gA within the short unique region of the viral genome on BamHI fragments 7 and 12. Additional polypeptides encoded by this region were characterized by their electrophoretic mobility. In three virus strains tested a similar, but strain-specific, pattern of the two gA precursors was found which was not dependent on the host cell or the state of infection after reaching the late phase.     

Location of the structural genes for glycoproteins gD and gE and for other polypeptides in the S component of herpes simplex virus type 1 DNA.

To map the structural genes for the gD and gE polypeptides and for other viral products encoded in the S component of herpes simplex virus type 1 DNA, we selected mRNAs capable of hybridizing to cloned viral DNA fragments and translated the mRNAs in vitro to determine which polypeptides were encoded therein. The gD and gE polypeptides were identified by immunoprecipitation with appropriate monoclonal and monospecific antibodies, whereas the other polypeptides were characterized only by their electrophoretic mobilities in polyacrylamide gels. We found that gD mRNA hybridized to a single SacI subfragment of BamHI fragment J, whereas gE mRNA hybridized to an adjacent SacI subfragment of BamHI fragment J and also to BamHI fragment X. These and other results permit the conclusion that the structural gene for gD is located between map coordinates 0.911 and 0.924, and the gene for gE is between map coordinates 0.924 and 0.951. We also found that mRNAs for polypeptides of 55,000, 42,000, 33,000, and 22,000 molecular weight hybridized to DNA fragments spanning the regions from map coordinates 0.911 to 0.924, 0.897 to 0.911, 0.939 to 0.965, and 0.939 to 0.965, respectively. Finally, in accord with the results of others, we found that mRNA for a 68,000-molecular-weight polypeptide hybridized to the two noncontiguous BamHI fragments N and Z, which share a reiterated DNA sequence.     

In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus Early and Late mRNAs

A preliminary translational map of the Autographa californica genome was constructed. Eighteen viral DNA restriction fragments were either purified from agarose gels or obtained from pBR322 recombinant DNA plasmids to locate specific gene products. The DNAs were immobilized on nitrocellulose filters and used to select viral mRNAs isolated from RNA obtained from the cytoplasm of infected Spodoptera frugiperda cells at 21 h postinfection. The fragment-specific mRNAs were translated in vitro in the presence of l-[(3)H]leucine by using a rabbit reticulocyte lysate system and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The approximate locations of 19 A. californica nuclear polyhedrosis virus (AcMNPV) gene products were mapped. The genes for mRNAs present late in viral infection were mapped to DNA fragments that represent nearly the entire genome. The molecular weights of many of these proteins were similar to those present in purified AcMNPV extracellular virus and to proteins being made in infected cells at 18 to 21 h postinfection. Cytoplasmic RNA was isolated at 4 h postinfection from infected cells, a time early in the viral infection cycle, and hybridized to AcMNPV DNA immobilized on nitrocellulose filters. AcMNPV-specific early RNA was translated in vitro into at least six polypeptides, the most abundant having a molecular weight of 39,000. Viral polypeptides were detected in cells pulse-labeled with l-[(3)H]leucine at 3 to 6 h postinfection, with molecular weights similar to those of polypeptides made in vitro from early AcMNPV mRNA.     

Mapping of herpesvirus saimiri proteins on the viral genome: proteins dependent and not dependent on viral DNA synthesis.

Hybrid selected translation was used to map the genome of herpesvirus saimiri, a lymphotropic and oncogenic herpesvirus. RNA extracted from virus-infected cells was hybridized to cloned genomic fragments, and the hybrid selected mRNAs were translated in vitro in a rabbit reticulocyte lysate. Forty-five virus-induced polypeptides were identified and correlated to their coding regions on the herpesvirus saimiri genome. Inhibition of the replication of viral DNA with phosphonoacetic acid showed that 22 of these polypeptides belong to the early group of herpesvirus saimiri gene products.     

Shotgun phage display — Selection for bacterial receptins or other exported proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins. Published: May 1, 2003     

Molecular-biological study of vaccinia virus genome. II. Localization and nucleotide sequence of vaccinia virus genes coding for proteins 36K and 12K

Genes encoding virus-specific late proteins with molecular mass 36 kDa and 12 kDa were mapped in HindIII-P DNA fragment of vaccinia virus strain L-IVP by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. RNA origin site of the 36K protein was detected in HindIII-J fragment. Nucleotide sequences of these genes were determined. Amino acid sequences of the 36K and 12K polypeptides were compared with the protein bank PIR.     

Cloning and molecular characterization of the HgiCI restriction/modification system from Herpetosiphon giganteus Hpg9 reveals high similarity to BanI.

The genes coding for the GGYRCC specific restriction/modification system HgiCI from Herpetosiphon giganteus Hpg9 have been cloned in Escherichia coli in three steps. As an initial step, the methyltransferase gene could be obtained after heterologous in vitro selection of a plasmid gene bank by cleavage with the isoschizomeric restriction endonuclease BanI. The adjacent endonuclease gene was cloned following Southern blot analysis of flanking genomic regions. The two genes code for polypeptides of 420 amino acids (M.HgiCI) and 345 amino acids (R.HgiCI). Establishing a functional endonuclease gene could only be achieved using a tightly regulated expression system or by methylation of the genomic DNA prior to transformation of the endonuclease gene. The methyltransferase M.HgiCI shows significant similarities to the family of 5-methylcytidine methyltransferases. Striking similarities could be found with both the isoschizomeric endonuclease and methyltransferase of the BanI restriction/modification system from Bacillus aneurinolyticus.     

Direct cloning by covalent attachment of probe DNA to target DNA.

A novel cloning procedure which makes use of covalent attachment of probe DNA to specific target DNA is reported. We show that specific gene fragments found in complex genomes such as the human genome can be cloned directly from a pool of genomic DNA with very high efficiency. This direct cloning method totally eliminates certain steps in current cloning procedures such as construction of DNA libraries and colony (plaque) hybridization. The resulting process has made cloning methods simpler and more time efficient, while achieving high cloning efficiency due to the stable nature of the probe-target DNA complex through covalent bonding. Most importantly, since clones are directly obtained from a pool of genomic DNA, the isolated clones are considered to be faithful copies of the original genes. This has apparently solved the problem of isolating clones with misincorporated bases or chimeric DNA, both of which are often encountered in cloning processes using PCR or other methods involving in vitro DNA synthesis.     

DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA.

DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight.     

Polycistronic mRNAs code for polypeptides of the Vibrio harveyi luminescence system.

DNA coding for the alpha and beta subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the alpha subunit as a hybridization probe, we identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the alpha and beta subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to and released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase alpha and beta subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.