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Biomedical subjects

M L Pearson

Publications and source records attributed to M L Pearson.

At least 55 records · Page 3Linked to original sources
PubMed1987

Aberrant regulation of synthesis and degradation of viral proteins in coliphage lambda-infected UV-irradiated cells and in minicells.

The patterns of bacteriophage lambda proteins synthesized in UV-irradiated Escherichia coli cells and in anucleate minicells are significantly different; both systems exhibit aberrations of regulation in lambda gene expression. In unirradiated cells or cells irradiated with low UV doses (less than 600 J/m2), regulation of lambda protein synthesis is controlled by the regulatory proteins CI, N, CII, CIII, Cro, and Q. As the UV dose increases, activation of transcription of the cI, rexA, and int genes by CII and CIII proteins fails to occur and early protein synthesis, normally inhibited by the action of Cro, continues. After high UV doses (greater than 2,000 J/m2), late lambda protein synthesis does not occur. Progression through the sequence of regulatory steps in lambda gene expression is slower in infected minicells. In minicells, there is no detectable cII- and cIII-dependent synthesis of CI, RexA, or Int proteins and inhibition of early protein synthesis by Cro activity is always incomplete. The synthesis of early b region proteins is not subject to control by CI, N, or Cro proteins, and evidence is presented suggesting that, in minicells, transcription of the early b region is initiated at a promoter(s) within the b region. Proteolytic cleavage of the regulatory proteins O and N and of the capsid proteins C, B, and Nu3 is much reduced in infected minicells. Exposure of minicells to very high UV doses before infection does not completely inhibit late lambda protein synthesis.

Bacteriophage lambda↗
PubMed1985

Polymorphism of the 3' open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III.

The genome of the virus associated with the acquired immune deficiency syndrome (AIDS), human T-lymphotropic virus type III (HTLV-III), includes two open reading frames, not found in other retroviruses. One of these, designated 3' open reading frame (3'orf) is 648 base pairs (bp) in length, and overlaps with the 3' long terminal repeat (LTR) sequences. Sequences of additional HTLV-III clones were determined in order to estimate the level and location of variation within 3'orf, to gain some insight into the function of its protein product. Newly determined sequences are reported for 3'orf of two unintegrated clones of HTLV-III and three cDNA clones made from virion RNA derived from the same cell line infected with pooled blood samples of different patients with AIDS or AIDS-related complex symptoms (ARC). In addition, sequences for 3'orf were derived from an unintegrated viral clone derived from a different cell line infected with a distinct isolate from a single patient. These sequences are compared to those previously reported for six other viral clones. Sequences of 3'orf differ among clones by 1.1-10.4% bp and 2.4-17.0% of predicted amino acids. This represents significantly greater sequence variation than is found in the entire genome on average. Moreover, a functional proviral clone has a termination codon at amino acid residue 124 of this open reading frame. This raises questions concerning the structure, and regulation of expression of the protein encoded by 3'orf.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Analysis of human blood monocyte activation at the level of gene expression. Expression of alpha interferon genes during activation of human monocytes by poly IC/LC and muramyl dipeptide.

Human monocytes were activated to secrete alpha interferon (IFN-alpha) by poly IC/LC but not by other monocyte activators, such as muramyl dipeptide (MDP). In contrast, monocytes were activated to secrete fibroblast growth factor (FGF) release by MDP but not by poly IC/LC. The amount of total RNA present in unactivated and activated human monocytes was similar. Using two 32P-labeled cDNA probes (pLM001 and HuIFN-alpha 2) for human IFN-alpha genes in hybridization studies, we analyzed messenger RNA species from this gene family in activated human monocytes. After activation with poly IC/LC, two other mRNA species (2.8 and 5.5 kb) were detected in addition to the 1.0 kb mRNA normally associated with IFN-alpha secretion. Unexpectedly, monocytes activated with MDP also contained 2.8 kb IFN-alpha mRNA. There was associated with this 2.8 kb IFN-alpha mRNA, found in MDP-activated monocytes, appreciable levels of intracellular IFN-alpha activity in the absence of detectable secreted IFN-alpha. Thus the secretion of IFN-alpha in activated human monocytes can be correlated with the appearance of a 1.0 kb mRNA species after poly IC/LC exposure. Secretion appears to be defective in MDP-stimulated monocytes even though they contain active intracellular IFN-alpha apparently translated from the 2.8 kb mRNA.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1985

Expression of a rat brain creatine kinase-beta-galactosidase fusion protein in Escherichia coli and derivation of the complete amino acid sequence of rat brain creatine kinase.

Expression of a rat brain creatine kinase (CKB)/beta-galactosidase fusion protein in Escherichia coli has allowed isolation of a rat CKB cDNA clone by direct antibody screening of a rat brain gamma gt11 expression library. This clone is 1416 bp long and includes 202 bp of 3'-untranslated region and 29 bp of 5'-untranslated region. The coding sequence of this clone has enabled us to deduce the complete amino acid (aa) sequence of rat CKB protein.

Amino Acid Sequence↗
PubMed1984

Isolation and sequence analysis of cDNA clones coding for rat skeletal muscle creatine kinase.

A series of cDNA clones corresponding to 1494 bases of rat muscle creatine kinase mRNA has been isolated and characterized. The identity of these clones has been confirmed by DNA sequence analysis and by comparison of the predicted amino acid sequence with that determined for the purified protein. The cDNA sequence accounts for the entire coding sequence of the creatine kinase protein in addition to the complete 3' untranslated region and 68 bases of 5' noncoding region. Sequences corresponding to the active site region of the protein, the initiation codon, the termination codon, and poly(A) addition signal have been identified.

Amino Acid Sequence↗
PubMed1983

Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

Animals↗
PubMed1983

Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

To assess the functional role of RNA polymerase II in the regulation of transcription during muscle differentiation, we isolated and characterized a large number of independent alpha-amanitin-resistant (AmaR) mutants of L6 rat myoblasts that express both wild-type and altered RNA polymerase II activities. We also examined their myogenic (Myo) phenotype by determining their ability to develop into mature myotubes, to express elevated levels of muscle creatine kinase, and to synthesize muscle-characteristic proteins as detected by two-dimensional polyacrylamide gel electrophoresis. We found a two- to threefold increase in the frequency of clones with a myogenic-defective phenotype in the AmaR (RNA polymerase II) mutants as compared to control ethyl methane sulfonate-induced, 6-thioguanine-resistant (hypoxanthine, guanine phosphoribosyl transferase) mutants or to unselected survivors also exposed to ethyl methane sulfonate. Subsequent analysis showed that about half of these myogenic-defective AmaR mutants had a conditional Myo(ama) phenotype; when cultured in the presence of amanitin, they exhibited a Myo- phenotype; in its absence they exhibited a Myo+ phenotype. This conditional Myo(ama) phenotype is presumably caused by the inactivation by amanitin of the wild-type amanitin-sensitive RNA polymerase II activity and the subsequent rise in the level of mutant amanitin-resistant RNA polymerase II activity. In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo- phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation.

Amanitins↗
PubMed1981

Enhancing the efficiency of DNA-mediated gene transfer in mammalian cells.

We have investigated several of the experimental factors that affect calcium phosphate-DNA-mediated gene transfer of thymidine kinase (tk) into mouse LM tk- Cl 1D cells using unfractionated DNA from both Chinese hamster ovary cells and L6 rat myoblasts. Increases in the length of exposure to DNA (24 h) and the expression time (48 h) before selection result in a 20-fold enhancement in the efficiency of transformation. These modifications yield frequencies up to 35 HATR colonies/20 microgram tk"NA/10(6) recipient cells. Exposure to dimethyl sulfoxide enhances transformation efficiencies slightly for short DNA exposure times, but has no effect when optimal DNA exposure times are used. Several other variations in our standard transformation protocol were also examined: these include the concentration and size of the DNA and exposure to low concentrations of the nonionic detergent, Tween-80. We have also isolated and characterized a subclone of Cl 1D that is a high-efficiency recipient for the tk+ marker. Segregation analysis reveals that the majority of the TK+ transformants derived from this subclone are stable, in contrast to those derived from the DL 1D parent. The combination of improved methodology and the high-efficiency recipient subclone permits DNA-mediated transformation for tk at frequencies on the order of 10(-4) transformants per recipient cell.

Animals↗
PubMed1981

Competence for DNA transfer of ouabain resistance and thymidine kinase: clonal variation in mouse L-cell recipients.

We have used the calcium phosphate precipitation technique to study the competence of mammalian cell recipients for transformation with genomic mammalian cell DNA. The transformation efficiency for thymidine kinase (tk) varies 10- to 20-fold (up to 10(-4) transformants/recipient) among different subclones of the LM tk- CL 1D mouse fibroblast cell line. Analysis of this phenotype among second-generation subclones indicates that subclones exhibiting high competence tend to breed true, whereas those with low competence do not. Isolation of Tk- revertants from TK+ transformants results in the selection of cells with a high-competence phenotype as measured by their subsequent transformation for tk. This phenotype appears to be a general characteristic of such cells because recipients more competent for transfer of a second marker, ouabain resistance (ouaR). This codominant marker coding for the Na K+-ATPase can be transferred at frequencies of 10(-5) in the high-competence recipients. These results indicate that competence for DNA-mediated gene transfer can be determined in part by genetic factors.

Animals↗
PubMed1981

Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability.

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cII protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cII protein caused by its decreased stability, since cII product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (lambda c1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion+ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.

Bacterial Proteins↗
PubMed1981

Downstream regulation of int gene expression by the b2 region in phage lambda.

Expression of the int gene after phage lambda infection normally requires the products of genes cII and cIII. However, when the phage carries a deletion in the nonessential b2 region adjacent to int, efficient synthesis of active Int protein does not require cII and cIII function. This inhibition of Int synthesis by nucleotide sequences downstream from the int structural gene behaves in a cis-dominant fashion in mixed infections. It is specific for PL- and not pI-initiated transcripts. Based on these observations, and those of others, a model is proposed in which Int translation from the pL transcript is inhibited by the interaction of downstream b2 nucleotide sequences and nucleotide sequences in the int region. The data imply a novel temporal mechanism regulating prophage lambda induction: circularization of the prophage genome results in the transposition of inhibitory b2 region sequences next to int and blocks further Int protein synthesis beyond the low level required for excision. As a consequence of this process, the control of int expression is transferred from the pL promoter to pI and the cII/cIII system. Such a genetic regulatory mechanism involving the rearrangement of genetic elements downstream from a structural gene may be of general use during development in other systems.

Bacteriophage lambda↗
PubMed1978

Identification of the N gene protein of bacteriophage lambda.

The N gene protein, pN, of bacteriophage lambda stimulates early gene transcription by allowing mRNA chain elongation to proceed into genes distal to transcription termination sites normally recognized by the Escherichia coli transcription termination protein rho. pN has previously eluded detection on sodium dodecyl sulfate/polyacrylamide gels because of its small size, its instability, and the difficulty of distinguishing pN itself both from host proteins and from other early lambda proteins whose synthesis depends on pN action. These problems have now been overcome and we find that the major form of pN present in crude cell extracts of infected cells has an apparent molecular weight of 13,500. lambdabio256, a deletion-substitution mutant terminating in N, codes for a shorter pN of molecular weight 12,500. A nonsense fragment of 10,500 molecular weight coded by lambdaN(am7) has also been identified. These conclusions are based on examination of the electrophoretic profiles of the proteins synthesized after infection of UV-irradiated E. coli by various lambdaN(-) temperature-sensitive, nonsense, and deletion-substitution mutants. It has also been possible to distinguish pN itself from other early lambda polypeptides by infecting ron(-) cells with either lambdaN(mar) phage allowing pN synthesis but not pN action or lambdaN(am) phage defective in pN synthesis and pN action. Our results together with previous data are discussed with respect to the possible existence of multiple molecular weight forms of pN and the location of the coding sequences in the N gene region.

Coliphages↗
PubMed1978

Mass fragmentographic determination of pentachlorophenol in rainbow trout.

Pentachlorophenol (PCP) is extracted from selected tissues of rainbow trout and converted to pentachloroanisole (PCA) by means of alkylation in the presence of potassium carbonate as a condensing agent. Pentachlorophenetole (PPT) is used as an internal standard. The electron impact mass fragmentography is carried out by monitoring m/e 280 (PCA), 294 (PPT), and 237. A plot of peak height vs. concentration is linear from 0.03 to 4.0 ng. Accuracy and precision of the method is +/-2% (N - 36). The results are accurate enough to warrant the use of this method for determining PCP in fish, as well as for monitoring PCP from various environmental sources. In addition, this technique provides a simple and rapid method by which a mixture of PCA and PCP can be determined simultaneously.

Animals↗