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Barnacles are marine sessile crustacean inhabiting intertidal areas of the Selangor coastline. They are seen attaching themselves to rocks and artificial structures such as jetty, piers, boats and sea walls. Being the most successful biofoulers, barnacles cause economic losses to some extent. Most of barnacles study focused on morphological identification only. Since molecular method gave more accurate results by sequence comparison, species identification was done on samples of obviously different species inhabiting artificial substrata by using mitochondrial 16S rDNA identification. In Kuala Selangor, there was only one species found on artificial substrata in Bagan Pasir and Pasir Penambang which was identified as Amphibalanus cirratus. Two species that differed in their morphological characteristics found on Morib sea walls were identified as Amphibalanus cirratus and Chthamalus malayensis. Phylogenetic tree based on the 16S rDNA showed that all the samples were in the same cluster reflecting that they are in the same clade.

The vaccinia virus gene encoding the 87-kDa protein that comprises the large subunit of ribonucleotide reductase (vvR1) was cloned into a bacterial expression vector under the control of an inducible promoter. Culture of Escherichia coli cells harboring the recombinant plasmid under standard induction conditions (0.4 mM isopropyl beta-D-thiogalactopyranoside, 37 degrees C) resulted in synthesis of a completely insoluble product. Production of soluble vvR1 was achieved by growing bacteria at low temperature (15 degrees C) during the induction period, initiating induction at low cell density, and using a low concentration (0.05 mM) of the inducer isopropyl beta-D-thiogalactopyranoside. Hydroxyurea, an inhibitor of ribonucleotide reductase, increased production of soluble vvR1 in a dose-dependent manner. Recombinant vvR1 was purified from a high salt extract of the E. coli lysate in four steps, the last utilizing an affinity column consisting of the carboxyl-terminal seven amino acids of the vvR2 protein linked to an insoluble resin. Using purified recombinant vvR2 to reconstitute active enzyme, we determined that maximizing the rate of CDP reduction required pH 8.0-8.8, 50 mM dithiothreitol, and 2 mM ATP. Specific activity of purified vvR1 was 122 nmol/min/mg. Limited proteolysis of the vvR1 protein revealed protease-resistant fragments approximately 30 and 58 kDa in size. To our knowledge, this study represents the first expression, solubilization, and isolation of a recombinant "eukaryotic" form of ribonucleotide reductase large subunit.

Ribonucleotide reductase has been suggested as a rate-limiting enzyme in DNA synthesis, partly because activities of the enzyme in cell-free preparations are low relative to rates needed to sustain DNA replication at observed rates. Vaccinia virus, with a large duplex DNA genome, encodes both subunits of a specific ribonucleoside diphosphate reductase. In this report, we describe quantitative analysis of ribonucleotide reductase protein levels and DNA accumulation in vaccinia virus-infected cell extracts, to correlate the supply of deoxyribonucleotides with the demand for these precursors in viral DNA synthesis. To do this, we generated polyclonal antisera to TrpE fusion proteins constructed from the carboxyl termini of both subunits of viral ribonucleotide reductase. We used S1 nuclease and immunoprecipitation analysis to determine the transcriptional and translational kinetics of vaccinia virus ribonucleotide reductase expression. Enzyme activity and ribonucleotide reductase protein stability were also assayed during the time course of viral infection. Enzyme-linked immunoassays were used to quantitate protein levels, and filter hybridizations were used to measure the accumulation of viral DNA. We show that ribonucleotide reductase activity in vaccinia virus-infected cells is severalfold higher than needed to provide deoxyribonucleotides at rates commensurate with DNA synthesis. Thus, while the enzyme is important as catalyst for the first committed reaction in DNA replication, it is not rate-limiting for this process.

The deoxyuridine triphosphatase gene of vaccinia virus, encoded by the open reading frame F2L, was cloned into Escherichia coli and expressed under the control of a bacteriophage T7 promoter. After induction of T7 RNA polymerase by isopropyl beta-D-thiogalactopyranoside, a 16.5-kDa peptide accumulated to high levels. This 16.5-kDa protein was purified to homogeneity and characterized. Gel filtration of the purified protein revealed a trimeric native structure. Biochemical analysis revealed the enzyme to be a metalloenzyme; enzymatic activity is inhibited by EDTA. This inhibition was reversed by the addition of Mg2+, Mn2+, or Zn2+. While the enzyme activity was highly specific for dUTP with an apparent Km of 0.94 microM, inhibition studies show that 8-azido-ATP acted as a competitive inhibitor of dUTP with a Ki of approximately 173 microM. Also, protection studies demonstrated that nucleotide competitors inhibit photoincorporation of the photoaffinity analogues [gamma-32P]5-azido-dUTP and [gamma-32P]8-azido-ATP. This suggests that while catalytic activity is limited to dUTP, other nucleotides can bind the active site.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.

The retroviral protease-encoding region, PR, situated between the gag and pol genes, underwent gene duplication in the lineage now represented by simian retrovirus type 1; the sequence of the duplicated segment has diverged considerably from the present PR sequence [Power, M.D., Marx, P.A., Bryant, M.L., Gardner, M.B., Barr, P.J. & Luciw, P.A. (1986) Science 231, 1567-1572]. The PR-like duplicated gene segment was at some point translocated to a new site within the pol gene of a lentivirus (subsequent to the divergence of human immunodeficiency virus type 1), where it has been maintained. We have identified in the vaccinia virus genome a sequence that is homologous to the PR-like duplicated gene segment of both types of retrovirus in an open reading frame whose product is predicted to be a 16.2-kDa protein. The vaccinia PR-like gene is located in the HindIII F fragment, and its product displays 31-34% amino acid identity to the two types of retroviral duplicated protease sequences over a region encompassing 125 amino acid residues. Sequences flanking the vaccinia gene showed no significant homology at either the DNA or amino acid level to the retroviruses. Nuclease S1 and primer extension analyses determined that the vaccinia gene is transcribed early in infection.

alpha-Amanitin-resistant vaccinia virus mutants were isolated after serial viral passages in BSC-40 cells that were carried out in the presence of inhibitory levels (6 micrograms/ml) of alpha-amanitin. One such mutant, alpha-27, was highly refractory (greater than 95%) to alpha-amanitin-mediated inhibition and was selected for further study. In the absence of drug, the phenotypes of alpha-27 and wild-type vaccinia virus were indistinguishable with respect to growth kinetics. DNA synthesis, protein synthesis, and morphogenesis. Infections in the presence of alpha-amanitin revealed two striking differences, however. First, wild-type virus was unable to catalyze proteolytic processing of the two major capsid proteins VP62 and VP60, whereas alpha-27 was most efficient at this process. Second, wild-type viral morphogenesis within the infected cells was arrested by alpha-amanitin at an apparently analogous step to that previously described for enucleated cells. This observation was supported by the ability of alpha-27 virus to replicate in enucleated BSC-40 cells. Restriction enzyme analyses of alpha-27 versus wild-type genomes revealed that a XhoI cleavage site was altered in the alpha-27 DNA molecule, suggesting a possible location for the alpha-amanitin resistance locus.

Amplification of the M2 gene encoding the small subunit of ribonucleotide reductase (EC 1.17.4.1) was analyzed in a collection of vaccinia virus (VV) isolates selected for resistance to 5 mM hydroxyurea (HU). Most of the mutants harbored tandem direct repeat arrays of the M2 gene, but several had duplicated M2 as an inverted repeat by genomic rearrangements involving the chromosomal termini. Novel joints formed by direct repeats were mapped, amplified in vitro, and sequenced. The junctions were simple fusions between DNA downstream and upstream of the M2 gene. Lack of sequence homology at the breakpoints indicated that the initial genomic rearrangements leading to gene amplification were due to nonhomologous recombination events.

The vaccinia virus gene that encodes the small subunit of ribonucleotide reductase was localized to the HindIII F fragment by using degenerate oligonucleotide probes. DNA sequencing revealed a leftward-reading open reading frame that predicted a protein of 37 kilodaltons whose amino acid sequence was much more homologous to the mouse and clam M2 sequences (approximately 80%) than to the corresponding herpesvirus (approximately 27%) or procaryotic (approximately 19%) gene products. Vaccinia virus mutants selected for the ability to grow in high concentrations of a specific inhibitor of ribonucleotide reductase, hydroxyurea, amplified the M2 gene and harbored tandem arrays (2 to 15 copies) of the gene within the HindIII F region. RNA isolated at early times after infection with wild-type virus and probed with an internal fragment of the M2 gene indicated one major (1.2 kilobases) and two minor (4.0 and 2.1 kilobases) transcripts. S1 nuclease analysis and primer extension experiments identified an RNA start site 12 nucleotides upstream of the putative initiation ATG codon.

A vaccinia virus (VV) gene required for DNA replication has been mapped to the left side of the 16-kilobase (kb) VV HindIII D DNA fragment by marker rescue of a DNA- temperature-sensitive mutant, ts17, using cloned fragments of the viral genome. The region of VV DNA containing the ts17 locus (3.6 kb) was sequenced. This nucleotide sequence contains one complete open reading frame (ORF) and two incomplete ORFs reading from left to right. Analysis of this region at early times revealed that transcription from the incomplete upstream ORF terminates coincidentally with the complete ORF encoding the ts17 gene product, which is directly downstream. The predicted proteins encoded by this region correlate well with polypeptides mapped by in vitro translation of hybrid-selected early mRNA. The nucleotide sequences of a 1.3-kb BglII fragment derived from ts17 and from two ts17 revertants were also determined, and the nature of the ts17 mutation was identified. S1 nuclease protection studies were carried out to determine the 5' and 3' ends of the transcripts and to examine the kinetics of expression of the ts17 gene during viral infection. The ts17 transcript is present at both early and late times postinfection, indicating that this gene is constitutively expressed. Surprisingly, the transcriptional start throughout infection occurs at the proposed late regulatory element TAA, which immediately precedes the putative initiation codon ATG. Although the biological activity of the ts17-encoded polypeptide was not identified, it was noted that in ts17-infected cells, expression of a nonlinked VV immediate-early gene (thymidine kinase) was deregulated at the nonpermissive temperature. This result may indicate that the ts17 gene product is functionally required at an early step of the VV replicative cycle.

We have developed a database of lymphoid polypeptides detected by two-dimensional polyacrylamide gel electrophoresis to aid in studies of leukemogenesis and of mutation affecting protein structure. In prior studies, we observed a 19-kDa phosphopolypeptide which was induced with proliferation in mature T cells and constitutively expressed in immature thymocytes. In this report we describe the identification of this polypeptide as the phosphorylated form of dUTPase (EC 3.6.1.23), following cDNA cloning of the gene, based on a partial amino acid sequence of the phosphopolypeptide. Studies of the expression and phosphorylation of dUTPase in human T cells indicate that accumulation and phosphorylation of dUTPase in mature T cells occur in a cell cycle-dependent manner. Interestingly, noncycling immature thymocytes express constitutively high levels of phosphorylated and unphosphorylated dUTPase. These results suggest an important role for dUTPase in immature thymocytes that is independent of proliferation.