Kenneth L. Roy

Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-β1 mRNA and protein than normal skin and cells

Transforming growth factor‐β1 is a well‐known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor‐β1 gene expression was evaluated in small skin samples. Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription‐polymerase chain reaction technique was used. Quantitation of gene expression by reverse transcription‐polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards. We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor‐β1 starting with a gene specific polymerase chain reaction product. After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen α1(I) cDNA with compatible ends was inserted into the polymerase chain reaction‐DNA fragment. The recombinant cDNA was re‐amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters. Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription‐polymerase chain reaction together with the tissue or cellular RNA. After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide. The bands for internal standard and transforming growth factor‐β1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription‐polymerase chain reaction. The number of mRNA molecules/cell was calculated. We examined the transforming growth factor‐β1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five‐fold more transforming growth factor‐β1 mRNA than normal skin per unit of wet weight. We used this procedure to quantitate transforming growth factor‐β1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin. The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor‐β1 than normal cells (116 ± 6 vs. 97 ± 7, p = 0.017, n = 5). These results were supported by Northern analysis for transforming growth factor‐β1 mRNA in the cells and enzyme‐linked immunosorbent assay for TGF‐β1 protein in fibroblast‐conditioned medium. In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor‐β1, which may be important in hypertrophic scar formation. The construction of the gene specific internal standard for reverse transcription‐polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.

Characteristics and genetic determinant of a hydrophobic peptide bacteriocin, carnobacteriocin A, produced by Carnobacterium piscicola LV17A.

Carnobacteriocin A is a hydrophobic nonlantibiotic bacteriocin that is detected early in the growth cycle of Carnobacterium piscicola LV17A and encoded by a 49 MDa plasmid. The bacteriocin was purified using hydrophobic interaction and gel filtration chromatography, and reversed-phase HPLC. Three different active peaks (A1, A2 and A3) were detected, but the purified samples had identical N-terminal amino acid sequences for the first 15 amino acids as determined by Edman degradation analysis. Only a 2.4 kb fragment of the EcoRI digest of the plasmid pCP49 hybridized with a 23-mer oligonucleotide probe derived from amino acids 5 to 13 of the amino acid sequence. The structural gene for carnobacteriocin A is located 600 base pairs into the 2.4 kb EcoRI fragment, but no other genetic information was detected on this unit. The structural gene includes an 18 amino acid N-terminal extension of the bacteriocin, ending with Gly-Gly residues in the -2, -1 positions with respect to the cleavage site. The bacteriocin consists of 53 amino acids that differ markedly from the majority of hydrophobic peptide bacteriocins characterized to date. Based on the amino acid sequence derived from the nucleotide sequence a molecular mass of 5052.85 Da was calculated. Mass spectrometric analysis showed that the molecular mass of the major component (A3) was 2 Da lower, thereby indicating the presence of a disulphide bridge between Cys 22 and Cys 51. Carnobacteriocin A2 has a similar structure except that Met 52 is oxidized to a sulphoxide, whereas A1 appears to be a mixture of peptides derived proteolytically from A3 or A2.

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus

SummaryA linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5′ termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.

Plasmid Transformation of Azotobacter vinelandii OP

Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron- and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42 degrees C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.

Two human tyrosine tRNA genes contain introns

A human lambda-phage recombinant which contains at least four tRNA genes, has been isolated. Two DNA fragments were subcloned to give the recombinant plasmids pM6 and pM6128. Nucleotide sequence analysis showed that each plasmid contained a different tyrosine acceptor tRNA (tRNATyr) gene. Both tRNATyr genes are interrupted by 21-bp introns. These recombinant plasmids have been shown to direct the in vitro synthesis of tRNA-sized products in a HeLa cell transcription system.

Transcriptional analysis of the isopenicillin N synthase-encoding gene of Streptomyces clavuligerus.

The gene (pcbC) encoding isopenicillin N synthase of Streptomyces clavuligerus is separated from an upstream open reading frame (ORF) by a 31-bp intergenic region. Inspection of the sequence of this intergenic region did not identify a promoter sequence. The promoter probe plasmid, pIJ4083, which contains the promoter-less catechol-2,3-dioxygenase (C23O)-encoding gene (xylE) as a reporter gene, was used to analyze the sequence upstream from the pcbC gene for promoter activity. Introduction of an SphI site at the start codon of pcbC by site-directed mutagenesis allowed the cloning of a 335-bp fragment (-334 to +1 in relation to the pcbC start codon) immediately upstream from xylE in pIJ4083. C23O activity was detected in both Streptomyces lividans and S. clavuligerus cultures that contained the upstream fragment, suggesting the presence of a promoter sequence. Northern analysis of total RNA extracted from S. clavuligerus identified a monocistronic 1.2-kb transcript hybridizing to a pcbC-specific probe. When RNA was isolated at various times during growth in liquid culture, the presence of a transcript was first detected during stationary phase. Analysis of the pcbC transcript by primer extension located the transcription start point to a C residue within the upstream ORF, 91 bp upstream from the pcbC start codon.

β-Lactamase of Lysobacter enzymogenes : cloning, characterization and expression of the gene and comparison of the enzyme to other lactamases

The gene for the periplasmic beta-lactamase of Lysobacter enzymogenes was isolated as part of a 1017 bp EcoRI fragment and the nucleotide sequence of the gene was determined. It has a G+C content of 71.5% and encodes a 27 amino acid signal sequence and the mature beta-lactamase of 276 amino acids which has a mass of 29,146 Da. The enzyme appears to be unique to L. enzymogenes but its amino acid sequence shows a high degree of homology with the amino acid sequences of the lactamase from Citrobacter diversus and other Class A beta-lactamases. The beta-lactamase gene of L. enzymogenes was expressed in Escherichia coli using pUC118 as the vector. The production of active beta-lactamase was highest after the active growth phase of the expression host and reached levels which were about three times higher than those obtained with L. enzymogenes.

A comparative study of the RNA-2 nucleotide sequences of two sweet clover necrotic mosaic virus strains

The nucleotide sequences of the RNA-2s of two strains of sweet clover necrotic mosaic virus (SCNMV-38 and -59) have been determined. The RNA-2s of SCNMV-38 and -59 consist of 1446 and 1449 nucleotides, respectively, and both contain one major open reading frame (ORF) which potentially can encode polypeptides of 326 amino acid residues (about 36.5K), designated SC38P2 and SC59P2, respectively. The nucleotide sequences of SCNMV-38 and -59 RNA-2s show 93.2% similarity, and the amino acid sequences of SC38P2 and SC59P2 are 91.7% identical, although the identical nucleotides and amino acids are not distributed uniformly in RNA-2 and the encoded proteins. Two highly conserved regions (from positions 23 to 221 and 297 to 326) and a relatively divergent region (from positions 222 to 296) are found in the P2 proteins of these strains. A similar pattern is apparent on comparison of the nucleotide and deduced amino acid sequences of RNA-2 of these SCNMV strains with those of the Australian and Czechoslovakian isolates of red clover necrotic mosaic virus.

The nucleotide sequence of turtle 5.8 S rRNA

The 5.8 S ribosomal RNA’s of human, rat and mouse cells have an essentially identical nucleotide sequence with minor differences due to terminal heterogeneity [ 1,2] . This sequence is 75% homologous with that of,yeast 5.8 S rRNA [3,4] but appears to be considerably less homologous with the analogous RNA from flowering plants [4,5]. In order to further examine the extent of homology among other eukaryotes and to detect evolutionary drifts, we have studied the 5.8 S RNA of turtle (Terrapene Carolina) heart cells and compared it to the presently known sequences. The results indicate that the nucleotide sequence of turtle 5.8 S rRNA differs from human cell RNA in only two respects: a single base substitution and a posttranscriptional modification.

Characterization of the tRNATrp genes of Saccharomyces cerevisiae

Abstract The purpose of this work was to examine the tRNA Trp -encoding genes ( tRNA Trp ) of Saccharomyces cerevisiae to gain insight as to why tRNA Trp amber suppressors, isolated by conventional genetic techniques, have not been reported. The results herein indicate that the haploid yeast genome contains six tRNA Trp genes which map to five or six chromosomes. Not only do the six genes have identical coding sequences but their introns are also identical. Gene replacement experiments indicate that five copies of tRNA Trp are sufficient for cell viability. Thus, mutation of one tRNA Trp gene to a suppressor in vivo, lowering the functional number of tRNA Trp genes, would not be expected to be lethal.