Transcriptional and posttranscriptional silencing of rodent alpha1(I) collagen by a homologous transcriptionally self-silenced transgene.

Transient transfection of rodent fibroblasts with plasmids carrying a full-size pro-alpha1(I) collagen gene (pWTC1) results in rapid reduction of the endogenous transcripts by >90%, while the transgene mRNA is undetectable. Using deletion constructs, two adjacent 5' noncoding regions of the gene are identified as causing transcriptional silencing of the endogene in normal and v-fos-transformed cells but not in nontumorigenic revertants, which show partial relief from v-fos transformation-induced alpha1(I) gene suppression. The 3' end of the transgene carries an additional element(s), causing posttranscriptional silencing of the endogene in all cells including the revertant. Data indicate that the transgenes are transcriptionally self-silenced. Genome-integrated transgenes that are transcriptionally active also allow expression of the endogene, suggesting gene activation by chromosomal factors missing in pWTC1. Silencing is not regulated by antisense RNA. Silencing of the endogenous pro-alpha1(I) collagen gene is not linked to the level of transgene expression.

Direct gene quantitation by PCR reveals differential accumulation of ectopic enzyme in rat-1 cells, v-fos transformants, and revertants.

Valid comparisons of gene promoter activities between different cell lines, and within a cell line, critically depend on accurate measurements of the number of genes introduced into the nuclei of cells. We have developed a simple method that allows direct and accurate quantitation of transfected plasmid DNA in cultured cells. The transfected DNA present in nuclei is copurified with genomic DNA without using phenol/chloroform extractions. DNA is amplified by PCR, and the amount of transfected DNA is read directly from a standard curve. By using the procedures described in this report, we have studied the relative expression of the Escherichia coli beta-galactosidase gene, driven by the wild-type Mo-MuLV LTR, in Rat-1 fibroblasts, FBJ v-fos-transformed Rat-1 (1302), and a revertant of v-fos-transformant (EMS-1-19) cell lines. The relative levels of expression of the transgene at 22 hr post-transfection in these three cell lines were 1:4:1, respectively, and at 48 hr post-transfection the respective ratios were 1:10.6:4. These results have significant implications for the use of cotransfected internal control plasmids to normalize data from transient transfection experiments to study promoter activities among different cell lines.

Molecular analysis of the effects of the lys 3a gene on the expression of Hor loci in developing endosperms of barley (Hordeum vulgare L.).

The lys 3a gene present in the barley mutant Ris phi 1508 results in an increased content of lysine in the grain. Previous studies have shown that this increase results from a decreased accumulation of hordein and an increase in other more lysine-rich proteins and in free amino acids. We report here a detailed examination of the effects of this gene on the different groups of hordein polypeptides and the mRNAs encoding them. The amounts of the two major groups of hordein polypeptides (B and C hordein ) were reduced to about 20 and 7%, respectively, of those present in the parental variety ( Bomi ), with a greater effect on one of the two subfamilies of B polypeptides. In contrast, the amounts of D hordein polypeptides were increased fourfold. In vitro translations of polysomal and total cellular RNA fractions showed similar effects on the relative amounts of hordein products synthesized. More detailed analyses of the populations of hordein mRNAs were made using specific cDNA clones and hybrid-selection translation, Northern hybridization, and "hybrid-dot" analysis. Only traces of mRNAs for "C" hordein were detected, while the abundances of mRNAs for the two subfamilies of B hordeins were reduced to 40 and 5% of those in Bomi . The amount of mRNA for D hordein was increased twofold. A cDNA clone related to B hordein was used to analyze genomic DNA fractions by Southern hybridization. The lys 3a gene had no effect on either the number (about 10) or the organization of the B hordein genes. These studies clearly demonstrate that the effects of the lys 3a gene on the amounts of the hordein polypeptides are closely related to changes in the amounts of the mRNAs encoding them. Although the exact effect of the gene remains unknown, it is most likely to be either at transcription or on the early processing of the mRNA.

Ribitol dehydrogenase messenger RNA from an enzyme superproducer strain of Klebsiella aerogenes. Purification, cell-free translation and studies in vitro and in vivo.

1. Ribitol dehydrogenase messenger RNA, from a strain of Klebsiella aerogenes that had been evolved to superproduce this enzyme, has been purified in a single step by labelling extracted polysomes with rabbit anti(ribitol dehydrogenase) and immunoprecipitating with sheep anti-(rabbit IgG). 2. The extracted mRNA is stable in a protein synthesis system in vitro and directs synthesis 35-40-times more efficiently than RNA from coliphages MS2 or Q beta, to give ribitol dehydrogenase as sole major product. 3. Its size distribution shows a major band of 1500 nucleotides plus fragments 400-1400 nucleotides, with only traces of size 2400-3000 nucleotides. Only the latter could encode both proteins of the operon: ribitol dehydrogenase and D-ribulokinase. 4. Ribitol dehydrogenase mRNA represents 24% of total mRNA in cells harvested just after a 'switch' point' in mid-exponential phase. About half of the polysomes containing this mRNA are unattached to DNA, whereas only 3% of other mRNAs are unattached to DNA. 5. This mRNA is not outstandingly stable in vivo, though there are indications that it may be more stable than average. Hence the high level of synthesis of ribitol dehydrogenase (up to 30% of total protein in an extract) seems to be due to very efficient transcription and translation from multiple copies of a constitutive rbtD gene.

Molecular cloning and analysis of cDNA sequences derived from poly A+ RNA from barley endosperm: identification of B hordein related clones.

A collection of over 130 cDNA clones has been constructed in the bacterial plasmids pPH207 and pBR322 using as template the poly A+ RNA from membrane-bound polysomes of barley endosperm (cv. Sundance). Fifty four B hordein cDNA clones have been identified by cross-hybridization analysis and in vitro translation of plasmid-selected mRNAs. Hybridization of 11 of the B hordein cDNA clones to Northern blots of size-fractionated RNA indicated that the B hordein mRNA is ca. 1300 nucleotides long. One cDNA clone, pHvE-c16, has been partially sequenced and shown by comparison with C-terminal and other peptide sequences to be related to B1 hordein polypeptides. The results obtained from the analysis of the B hordein cDNA clones support the idea that the Hor 2 locus, which specifies the B hordeins, is complex and codes for a family of related mRNA species.

A switch from translational control to transcriptional control of protein synthesis in mid-exponential growth phase of bacterial cultures. Specific radioimmune labelling of ribitol-dehydrogenase-synthesising polysomes from Klebsiella aerogenes in the presence of heparin.

1. We present evidence suggesting a sudden switch from translational control to transcriptional control of protein synthesis in mid-exponential growth of bacterial batch cultures. At a critical cell density a switch from large to small polysomes occurs during a short period of exponential growth. The profile of specific polysomes engaged in synthesis of a constitutive enzyme, ribitol dehydrogenase, changes at the same point but in an opposite way: a linear profile peaking at monosomes changes to a dome-shaped profile peaking at about 15 ribosomes/mRNA, which persists into late exponential phase despite a gradual reduction in the total polysome population. The switch in the pattern of protein synthesis is exhibited dramatically by changes in the specific activity or ribitol dehydrogenase in cell extracts at different stages of batch culture. In early exponential phase the specific activity of the enzyme is constant, but it begins to rise suddenly, at the same point at which the polysome profiles change, and continues to increase up to the end of exponential phase. This effect is exhibited by the strains of Klebsiella aerogenes that are inducible for (in the presence of the inducer), consitutive for, or superproducers of ribitol dehydrogenase, and it appears to be unrelated to catabolite repression. 2. The above results depend on improved techniques for production of large amounts of bacterial polysomes and the ability to label nascent peptides attached to polysomes very specifically with radioactive antibody to ribitol dehydrogenase. Our success was due to the observation that sodium heparin completely abolishes non-specific interactions of the antibody with the polysomes.

Reversal by aromatic amino acids of 2-thiazole-DL-alanine inhibition of Salmonella typhimurium.

Of 21 l-amino acids tested (at 1.2 x 10(-4)m), only histidine and the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) protect Salmonella typhimurium strains from inhibition of growth and immediately reverse the growth inhibition by 5 x 10(-4)m 2-thiazole-dl-alanine.