Biomedical subjects

D M Hinton

Publications and source records attributed to D M Hinton.

At least 19 recordsLinked to original sources

PubMed1992

Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage.

The bacteriophage T4 segA gene lies in a genetically unmapped region between the gene beta gt (beta-glucosyltransferase) and uvsX (recombination protein) and encodes a protein of 221 amino acids. We have found that the first 100 amino acids of the SegA protein are highly similar to the N termini of four other predicted T4 proteins, also of unknown function. Together these five proteins, SegA-E (similar to endonucleases of group I introns), contain regions of similarity to the endonuclease I-Tev I, which is encoded by the mobile group I intron of the T4 td gene, and to putative endonucleases of group I introns present in the mitochondria of Neurospora crassa, Podospora anserina, and Saccharomyces douglasii. Intron-encoded endonucleases are required for the movement (homing) of the intron DNA into an intronless gene, cutting at or near the site of intron insertion. Our in vitro assays indicate that SegA, like I-Tev I, is a Mg(2+)-dependent DNA endonuclease that has preferred sites for cutting. Unlike the I-Tev I gene, however, there is no evidence that segA (or the other seg genes) resides within introns. Thus, it is possible that segA encodes an endonuclease that is involved in the movement of the endonuclease-encoding DNA rather than in the homing of an intron.

Amino Acid Sequence↗

PubMed1992

Testing guidelines for evaluation of the immunotoxic potential of direct food additives.

Immunotoxicity testing is a new addition to the safety assessment guidelines for direct food and color additives. The approaches and philosophy for this area of specialized testing are consistent with the case-by-case strategy applied in the regulatory approval process, which is based on structure-activity relationships, preexisting knowledge, and projected exposure estimates. Specialized testing such as immunotoxicity is not part of the basic testing requirements, but would be applied when indicators are positive. Concepts for immunotoxicity testing have evolved, in part, from research for evaluating various testing methods as well as specific study designs. This research, conducted over the last decade, has focused mainly on the rat as the rodent species of choice. The miniature swine was evaluated as a nonrodent model. Testing is defined by type 1 and type 2 tests, which differ in that type 1 tests are performed on the same animals used in the core study design. Sets of type 1 and type 2 tests, with reference to the indicators, define various testing levels. Retrospective testing, expansion of basic testing (such as histopathology and serum chemistry profiles), and alternative study designs, which include satellite groups for evaluation of the functional capacity of the immune system, can be considered in the evaluation of immunotoxic potential.

Animals↗

PubMed1991

Transcription from a bacteriophage T4 middle promoter using T4 motA protein and phage-modified RNA polymerase.

The bacteriophage T4 motA protein is required for transcription from T4 middle promoters. These promoters, which contain the Escherichia coli promoter consensus sequence at the -10 region (TATAAT) but a unique sequence centered at -30 ((a/t)(a/t)TGCTT(t/c)A) (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258), become active about 2 min after infection, a time when the host RNA polymerase has been modified by phage proteins. This paper shows that motA protein binds to a T4 middle promoter in vitro and that the addition of the motA protein allows in vitro transcription from this promoter by T4-modified RNA polymerase. The T4 motA gene was cloned into a multicopy plasmid that complemented T4 motA mutants in vivo. MotA protein, partially purified from cells containing a motA+ plasmid, specifically retarded the electrophoretic mobility of an oligomer containing the T4 middle promoter located 195 bases upstream of uvsX (PuvsX). RNA polymerase isolated from infected cells during T4 middle gene expression supported in vitro transcription from PuvsX only when fractions containing the motA protein were added. In contrast, unmodified host RNA polymerase catalyzed the synthesis of minor amounts of RNA from PuvsX, but this synthesis was not motA dependent. Thus, the in vitro transcription system described here provides the basis for a detailed study of the phage and host factors needed to regulate T4 middle gene expression.

Base Sequence↗

PubMed1991

Accumulation of 2,8 dihydroxyadenine in bovine liver, kidneys, and lymph nodes.

A variety of tissues from 20 cattle slaughtered at federally inspected facilities contained abundant light green to greenish-yellow material. Gross lesions were most common in the liver and hepatic lymph nodes. Less frequent lesions were present in the mediastinal, renal, intercostal, and gastric lymph nodes. The material was most prominent in the portal triads, and in the medullary sinuses of the lymph nodes, at times occupying up to one half of the nodal mass. Renal calculi were present in one animal. Histologically, the condition was characterized by the intracytoplasmic accumulation of innumerable brown, acicular crystals in hepatocytes, macrophages, and renal tubular epithelial cells. Less frequent large aggregates of extracellular crystals were found in the lumens of renal tubules and in portal triads. Crystals were highly birefringent when examined using polarized light. The crystals were identified as 2,8 dihydroxyadenine using X-ray diffraction, electron diffraction, infrared spectroscopy, and mass spectrometry. In mammals, adenine is normally converted to adenylate by the enzyme adenine phosphoribosyltransferase. When adenine phosphoribosyltransferase is absent, deficient, or inhibited, adenine is oxidized to 2,8 dihydroxyadenine, which is extremely insoluble at physiological pH. In human beings, an autosomal recessive disease known as 2,8 dihydroxyadeninuria is caused by a deficiency of adenine phosphoribosyltransferase.

Abattoirs↗

PubMed1990

Subchronic oral toxicity of cyclopiazonic acid (CPA) in male Sprague-Dawley rats.

The mycotoxin cyclopiazonic acid (CPA) is a potential contaminant of processed foods, grain and poultry. Twelve male Sprague-Dawley rats were given oral doses of 0, 0.2, 0.6, 2.0 or 4.0 mg CPA/kg body weight/day for 13 consecutive weeks to study its potential subchronic toxicity. No dose-related mortality or morbidity occurred. General appearance, behavior, body weight gain and food consumption of all groups were similar. CPA had no definite adverse hematologic or serum chemistry effects, although serum creatinine concentrations of rats given 2.0 and 4.0 mg CPA/kg BW were increased after seven and 13 weeks. Mild to focally moderate acute inflammation of the lamina propria and submucosa of the gastric epithelium was found in animals given less than or equal to 0.6 mg CPA/kg BW. No other dose-related microscopic lesions were found. Ultrastructural examination of the livers revealed subtle disruption of the cisternal pattern of the endoplasmic reticulum with ribosomal detachment in animals receiving 4.0, but not 2.0, mg CPA/kg BW. These data suggest that the toxic effects in rats of repeated, daily oral exposure to CPA may be less than previously reported. The possible relationship between toxicity and CPA epimerization is considered.

Administration, Oral↗

PubMed1990

Chronology of patulin-induced alterations in membrane function of cultured renal cells, LLC-PK.

In a previous study we compared the effects of patulin (PAT) and ouabain, a specific inhibitor of the Na(+)-K+ ATPase, and found significant differences with regard to the kinetics of Na+ influx and K+ efflux, and sulfhydryl reactivity in LLC-PK1 cells. The purpose of the present study was to determine the relationship between Na+ influx, K+ efflux, membrane potential ([3H]tetraphenylphosphonium accumulation), cellular viability [lactate dehydrogenase (LDH) release], and changes in cell morphology (blebs). The effects of PAT are concentration and time dependent. At concentrations of PAT above 10 microM there is a transient increase in intracellular electronegativity (less than 1 hr) followed by a sustained depolarization (greater than 1 hr) which is correlated with complete Na+ influx, K+ efflux, total LDH release, and bleb formation. However, at PAT concentrations of 5-10 microM there is a sustained increased intracellular electronegativity (4-8 hr) which is associated with partial Na+ influx and K+ efflux, no significant LDH release, and relatively few blebs. The hyperpolarizing effect may be a result of increased permeability to K+ relative to Na+. At times and concentrations which result in increased intracellular electronegativity, PAT has no effect on [3H]ouabain binding and thus increased Na+/K+ pump turnover does not seem to be the cause of the transient hyperpolarizing effect of PAT. These results are consistent with the hypothesis that PAT causes alterations in plasma membrane permeability which favor K+ efflux relative to Na+ influx. The toxic effects of PAT are irreversible in LLC-PK1 cells after even short pretreatment with PAT. The primary toxic lesion appears to be at some level other than that involving inhibition of macromolecular synthesis, perhaps the plasma membrane itself.

Animals↗

PubMed1989

Transcript analyses of the uvsX-40-41 region of bacteriophage T4. Changes in the RNA as infection proceeds.

The bacteriophage T4 genes uvsX (recombination protein), 40 (stimulates head formation), and 41 (DNA replication protein, part of the primase-helicase) are located together on the T4 genome (5'----3' uvsX-40-41). Previous analyses have indicated that all three proteins are expressed within 5 min after infection and that the level of 41 protein is less than that of uvsX. The mapping of transcripts from this region (reported here) shows that this expression arises from polycistronic messages detected between 2-4 min after infection, a time when phage-encoded factors are beginning to alter the host transcriptional apparatus. Major RNA 5' ends, 900 and 200 bases upstream of uvsX, show homology with previously deduced T4 transcription sites dependent on the T4 transcription factor motA (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258). Analysis of the 3' end of uvsX RNAs shows that initially most transcripts extend through gene 40 and 41, although approximately equal to one-fourth end just past uvsX (within gene 40). Later, more of the uvsX messages are monocistronic, having 5' ends close to the gene (200 and 55 bases upstream) and having the 3' end within gene 40. Thus, during infection the level of 41 RNA is lowered relative to uvsX message. Mapping of RNA expressed from an uvsX-40-41 plasmid in an uninfected cell gives 5' ends 700, 450, and 55 bases upstream of uvsX, i.e. positions different from those during T4 infection. This indicates that infection significantly changes the 5' ends for uvsX RNA, either by altering transcription initiation or RNA processing sites. In contrast, the majority of the uvsX RNAs expressed by plasmid in the uninfected cell do end at the stop mapped during infection. Thus, the host alone can produce this 3' end.

Escherichia coli↗

PubMed1989

Altered expression of the bacteriophage T4 gene 41 (primase-helicase) in an Escherichia coli rho mutant.

Bacteriophage T4 gene 41 protein is an essential replication protein, part of the primase-helicase required for lagging strand DNA synthesis. In a T4+ infection, 41 RNA is first expressed as a polycistronic transcript attached to the upstream RNA of genes uvsX (recombination protein) and 40 (stimulates head formation (Hinton, D. M. (1989) J. Biol. Chem. 264, 14432-14439). As infection proceeds, less of the upstream RNA extends into gene 41 due to an RNA 3' end, approximately equal to 60 bases downstream of uvsX. DNA sequence analysis of this region positions this end within gene 40, immediately after a GC-rich hairpin. This end probably arises from host factor-dependent transcription termination or RNA processing since it is observed in RNA expressed by a uvsX-40-41 plasmid in vivo, but is not seen after in vitro transcription with purified Escherichia coli RNA polymerase. The E. coli transcription termination (rho) mutant rho026 has been characterized as a rho mutation whose terminating activity is not effectively overcome by phage lambda antitermination (Das, A., Gottesman, M. E., Wardwell, J., Trisler, P., and Gottesman, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5530-5534). During a T4+ abortive infection of rho026, the levels of some phage proteins, including 41, are depressed; a T4 phage mutant in goF gives wild type protein patterns in rho026 (Stitt, B. L., and Mosig, G. (1989) J. Bacteriol., in press). The RNA analyses presented here demonstrate that the severalfold decrease in 41 protein in rho026 is accompanied by a similar decrease in 41 RNA. There is both a general reduction in polycistronic uvsX-40-41 RNA and a 2-2.5-fold increase in the proportion of uvsX RNA ending at the 3' end. Infection of rho026 by T4 goF1 returns the relative amount of RNA reading into 41 versus that stopped to near a wild type level. These results suggest that host rho and the T4 goF are involved in the expression of T4 41 RNA.

Amino Acid Sequence↗

PubMed1989

Patulin-induced ion flux in cultured renal cells and reversal by dithiothreitol and glutathione: a scanning electron microscopy (SEM) X-ray microanalysis study.

Patulin (PAT), a compound produced by certain species of Aspergillus, Penicillium, and Byssochlamys, is frequently found associated with agricultural commodities. PAT has many effects on membrane function, including the inhibition of the isolated Na+-K+ ATPase. In this study, a scanning electron microscope equipped with an energy dispersive spectroscopy X-ray microanalysis system was used to examine individual cultured renal epithelial cells (LLC-PK1) in order to determine the effects of PAT on the relative intracellular ion concentrations. The estimated EC50 (60 min) for both sodium influx and potassium efflux was between 10 and 50 microns for ouabain. For PAT, the EC50 (60 min) was 250 microns for sodium influx and 100 microns for potassium efflux. However, 1 mM patulin at 240 min caused complete reversal of the sodium and potassium content of cells, and 1 mM ouabain at 240 min did not. The effect of patulin on sodium and potassium flux was both concentration and time dependent and was reversed by dithiothreitol and glutathione. PAT (250 microM) but not ouabain (250 microM) induced massive blebbing of LLC-PK1 cells. Thus, the interaction of PAT with cellular membranes involves both alterations in the regulation of intracellular ion content and the cytoskeleton. We hypothesize that patulin alters intracellular ion content via Na+-K+ ATPase and non-Na+-K+ ATPase mechanisms.

Animals↗

PubMed1989

Production of fusarin C on cereal and soybean by Fusarium moniliforme.

Two isolates of Fusarium moniliforme were compared with respect to production of a mutagenic compound, fusarin C, on seven corn varieties as well as on soybean, wheat, rye, barley, and a liquid culture medium. The isolates were originally obtained from corn and barley. Both isolates produced fusarin C on seed of all five crops within a 21-day period, and one isolate produced the largest amount on oats. Soybean was the poorest substrate for both isolates. Although the quantity of fusarin C produced on grain was isolate dependent, specific substrate requirements for each strain were suggested. The isolates differed in their ability to grow and produce fusarin C on corn with different moisture contents (16, 20, 24, and 28%). One isolate was more xerotolerant and grew at 16% moisture but did not produce the mutagen.

Culture Media↗

PubMed1987

Bacteriophage T4 DNA primase-helicase. Characterization of oligomer synthesis by T4 61 protein alone and in conjunction with T4 41 protein.

The bacteriophage T4 41 and 61 proteins function as a primase-helicase which in vitro both unwinds double-stranded DNA and synthesizes the pentaribonucleotides used to initiate DNA synthesis on the lagging strand. We demonstrate that 61 protein alone possesses a weak DNA template-dependent oligomer synthesizing activity, whose products differ in size and nucleotide specificity from those made by the 61 and 41 proteins together. We have previously shown that the 61 and 41 proteins make primarily ribonucleotide pentamers of the sequence pppApC(pN)3, although some pentamers beginning with G were also detected on phi X174 single-stranded DNA. The pentamers pppApC(pN)3 have also been shown to initiate T4 DNA chains in vivo (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861). We now show that in contrast, the major products made by 61 protein alone on phi X174 DNA with [alpha-32P]CTP and the other three ribonucleoside triphosphates are not pentamers, but the dimers pppApC and pppGpC. In addition, minor amounts of products from 3 to approximately 45 nucleotides in length are also synthesized. Unlike the 61/41 protein reaction, 61 protein alone can substitute dATP or dGTP for ATP or GTP. Addition of 41 protein greatly stimulates oligomer synthesis, especially the synthesis of products made with ATP and CTP and products 5 nucleotides in length. Thus, both 61 and 41 proteins are needed to obtain efficient synthesis of the biologically relevant pentamers pppApC(pN)3. We demonstrate that the glucosylated hydroxymethylcytosines present in T4 DNA do not support the initiation of primer synthesis by the 61 protein on this template. With glycosylated hydroxymethyl T4 DNA, pppApC but not pppGpC oligomers are detected. If the T4 DNA is modified by hydroxymethylation but not glucosylation, pppApC and only a trace of pppGpC products are seen. In the accompanying paper (Nossal, N.G., and Hinton, D.M. (1987) J. Biol. Chem. 262, 10879-10885), we examine DNA synthesis primed by 61 protein in the absence of 41 protein.

DNA Helicases↗

PubMed1987

Bacteriophage T4 DNA primase-helicase. Characterization of the DNA synthesis primed by T4 61 protein in the absence of T4 41 protein.

The bacteriophage T4 61/41 protein primase-helicase is part of a seven T4 protein system needed for DNA synthesis in vitro. Although both 41 and 61 proteins are required for the synthesis and utilization of the normal pppApC(pN)3 pentanucleotide primer, we show in the accompanying paper (Hinton, D. M., and Nossal, N. G. (1987) J. Biol. Chem. 262, 10873-10878) that high concentrations of 61 protein alone carry out a limited, template-dependent oligonucleotide synthesis with the dimers pppApC and pppGpC as the major products labeled with [alpha-32P]CTP. At these high concentrations, 61 protein alone primes DNA synthesis by T4 DNA polymerase and the T4 genes 44/62 and 45 polymerase accessory proteins, or by Escherichia coli DNA polymerase I. The addition of T4 replication proteins other than 41 protein does not change the size distribution of oligonucleotides made by 61 protein. However, the primers used for DNA synthesis in the absence of 41 protein are not dimers, but rather trace quantities of longer oligonucleotides (5 to about 45 bases) which begin predominantly with pppGpC. These results show that 41 protein is required to prime with oligonucleotides beginning with pppApC and suggest that 41 protein, either alone or in conjunction with 61 protein, helps to stabilize the usual short pentamer primers on the template until they are elongated by the DNA polymerase. Moreover, since 61 protein by itself can only initiate DNA synthesis with primers beginning with pppGpC, but cannot make oligonucleotides starting with pppGpC on T4 DNA in which all the C is glucosylated and hydroxymethylated, both the T4 41 and 61 proteins are essential to prime DNA synthesis on their normal template. In our analysis of RNA-primed DNA, we demonstrate that although RNA primers at the 5' ends of DNA chains are relatively resistant to the 3' to 5' exonuclease of T4 DNA polymerase (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861), pppNpNpNpNpN oligomers are digested to a greater extent than the dephosphorylated pentamers NpNpNpNpN.

Bacteriophage phi X 174↗

PubMed1987

Combined effects of the mycotoxins aflatoxin B1 and cyclopiazonic acid on Sprague-Dawley rats.

This study was conducted to determine whether exposure to cyclopiazonic acid (CPA) and aflatoxin B1 (AFB1) would alter the toxicity associated with exposure to either toxin individually. Groups of male rats were administered 0, 0.1 or 4.0 mg CPA/kg body weight/day intragastrically (three groups per dose level) for three consecutive days and 30 min after each of these CPA doses the rats were dosed by gavage with 0, 0.1 or 2.0 mg AFB1/kg body weight/day. Six of the 12 rats given each of these nine treatments were killed on day 4 after the initial dosing, and the rest were allowed a recovery period of 4 days prior to being killed. Weight loss in the three groups receiving 2.0 mg AFB1/kg/day occurred within 24 hr of the first doses. Feed consumption by these rats was about 60% of that in the other groups. By the end of the recovery period, rats in these three groups had lost an average of 31-38 g. Feed consumption throughout the recovery period by rats in the 2.0-mg AFB1 groups was about 50% of the control value, except in the group that also received the high dose of CPA, in which it was 75%. Gross pathological findings were primarily limited to rats in the high AFB1 group, and included icterus, shrunken liver and lesions in the kidney at the cortico-medullary junction. Microscopic changes were characteristic of aflatoxicosis in rats. Glycocholic acid assays indicated liver damage only in those groups that received the high AFB1 dose. We conclude that neither toxin potentiates the action of the other at the dose levels used in this study.

Aflatoxin B1↗

PubMed1987

Evaluation of immunotoxicity in a subchronic feeding study of triphenyl phosphate.

Triphenyl phosphate (TPP), a potential food contaminant, was fed to weanling Spartan Sprague-Dawley rats at dose levels of 0, 0.25, 0.5, 0.75, and 1.0% for 120 days. The immunotoxicity evaluation, planned as a minimum testing model in a subchronic study design as well as to provide information on TPP, was performed along with the routine testing of a separate group of animals. Traditional measures were made of growth and food consumption, total protein analysis, electrophoretic analyses of serum proteins, lymphoid organ weights in relation to growth, and histopathology, with expanded immunohistochemical evaluation of B- and T- lymphocyte regions in spleen, thymus, and lymph nodes, using immunoperoxidase staining. Assessment was made of the humoral response to a T-lymphocyte-dependent antigen, sheep red blood cells, and was begun at midterm of the feeding period for the primary response followed by secondary and tertiary booster immunizations at 3-week intervals. The kinetics of the responses were measured by hemolysin assay of relative antibody titers at days 3, 4, 5, and 6 postinjection. No significant effects on the responses were noted for either sex at any of the dose levels tested. The only effects noted were a decreased rate of growth at high levels of TPP and increases in the levels of alpha- and beta-globulins suggestive of increased hepatic activity.

Animals↗

PubMed1986

Cloning of the bacteriophage T4 uvsX gene and purification and characterization of the T4 uvsX recombination protein.

The bacteriophage T4 uvsX gene is a nonessential gene required for normal levels of DNA repair, recombination, and replication. We demonstrate that plasmids containing the T4 DNA approximately 300-2900 base pairs upstream of T4 gene 41 express a biologically active uvsX protein. This uvsX protein imparts increased survival to UV-irradiated T4 uvsX- phage and decreases the T4 uvsX- mutant suppression of a conditionally lethal T4 mutant in the gene 49 recombination nuclease. The uvsX protein purified from cells with a uvsX+ plasmid catalyzes ATP hydrolysis to ADP and AMP and, in the presence of the T4 gene 32 helix-destablizing protein, ATP-dependent strand exchange between homologous circular single-stranded and linear duplex DNA. These results agree with the recent characterization of uvsX protein from T4-infected cells by Yonesaki et al. (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H. (1985) Eur. J. Biochem. 148, 127-134) and by Formosa and Alberts (Formosa, T., and Alberts, B.M. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 363-370). In addition, we find that under some reaction conditions strand exchange is catalyzed by uvsX protein in the absence of 32 protein. The level of the uvsX protein expressed by the uvsX+ plasmids is high and independent of the orientation of the T4 DNA within the vector. This suggests that transcription promoter(s) lie upstream of the uvsX gene on the cloned T4 DNA. In vitro transcription of T4 DNA restriction fragments reveals two tandem promoters whose transcripts initiate approximately 500 and 600 nucleotides upstream of the uvsX gene and extend through the gene.

Adenosine Triphosphatases↗

PubMed1985

Bacteriophage T4 DNA replication protein 41. Cloning of the gene and purification of the expressed protein.

The bacteriophage T4 primase, composed of the T4 proteins 41 and 61, synthesizes pentaribonucleotides used to prime DNA synthesis on single-stranded DNA in vitro. 41 protein is also a DNA helicase that opens DNA in the same direction as the growing replication fork. Previously, Mattson et al. (Mattson, T., Van Houwe, G., Bolle, A., Selzer, G., and Epstein, R. (1977) Mol. Gen. Genet. 154, 319-326) located part of gene 41 on a 3400-base pair EcoRI fragment of T4 DNA (map units 24.3 to 21.15). In this paper, we report the cloning of T4 DNA representing map units 24.3 to 20.06 in a multicopy plasmid vector. Extracts of cells containing this plasmid complement gene 41- extracts in a DNA synthesis assay, indicating that this region contains all the information necessary for the expression of active 41 protein. We located gene 41 more precisely between T4 map units 22.01 to 20.06 since our cloning of this region downstream of the strong lambda promoter PL results in the production of active 41 protein at a level 100-fold greater than after T4 infection. We have purified 133 mg of homogeneous 41 protein from 27 g of these cells. Like the 41 protein from T4 infected cells, the purified 41 protein in conjunction with the T4 gene 61 priming protein catalyzes primer formation (assayed by RNA primer-dependent DNA synthesis with T4 polymerase, the genes 44/62 and 45 polymerase accessory proteins, and the gene 32 helix-destabilizing protein) and is a helicase whose activity is stimulated by T4 61 protein.

Ammonium Sulfate↗

PubMed1985

Bacteriophage T4 DNA replication protein 61. Cloning of the gene and purification of the expressed protein.

In vitro, a bacteriophage T4 primase composed of T4 61 and 41 proteins, catalyzes the formation of pentaribonucleotides used to initiate DNA synthesis on single-stranded DNA. We have determined that cells containing a plasmid with the T4 DNA from 18.68 to 15.05 map units express an activity that substitutes for authentic 61 protein in vitro in catalyzing primer-dependent DNA synthesis with six other T4 DNA replication proteins. This result establishes that this region, genetically assigned to gene 61, is the structural gene for the priming protein. Cells containing a plasmid with gene 61 downstream of the strong phage lambda promoter PL and the antitermination site nutL produce 100-fold more 61 protein than T4-infected cells. We have developed an improved purification procedure which yields 100 mg of homogeneous, active protein from 178 g of these cells. In the plasmid, the T4 DNA downstream of gene 61 expresses a protein of 30,000 daltons. This protein may be the T4 DNA adenine methylase (dam) gene product, since Schlagman and Hattman (Schlagman, S. L. and Hattman, S. (1983) Gene 22, 139-156) have shown that this activity is expressed by plasmids containing T4 DNA from this region. In the PL, nutL vector, the expression of both the 30,000-dalton and 61 proteins is enhanced up to 20-fold by the presence of the phage lambda N protein, a transcription antitermination protein, suggesting that expression of the T4 DNA in the plasmid may be regulated transcriptionally. In addition, in both N+ and N- cells, the level of 61 protein, whose gene is proximal to PL on the plasmid, is lower than that of the product of the promoter distal 30,000-dalton protein gene. This result suggests that, at least in the plasmid construction, the expression of 61 protein may also be regulated after transcription.

Bacteriophage lambda↗

PubMed1979

The synthesis of oligodeoxyribonucleotides using RNA ligase.

T4 RNA ligase catalyzes the addition of a single deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of oligodeoxyribonucleotides (Hinton et al. (1978) Biochemistry 17, 5091). We have determined improved conditions for this reaction which give yields equal to or greater than 85% when any of five common deoxyribonucleoside bisphosphate (pdAp, pdCp, pdGp, pdTp, or pdUp) are added to dA(PDA)4. A low ATP concentration, which is constantly maintained by a regeneration system composed of phosphocreatine, creatine kinase, and myokinase, contributes to the attainment of high yields. The addition of RNase A and spermine also enhances the rates and yields of the reactions. These conditions facilitate the use of RNA ligase as a reagent for the stepwise synthesis of DNA of defined sequence.

Base Sequence↗