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P J Farnham

Publications and source records attributed to P J Farnham.

63 records · Page 4Linked to original sources
PubMed1985

Opposite-strand RNAs from the 5' flanking region of the mouse dihydrofolate reductase gene.

We have characterized a transcription unit in the 5' flanking region of the mouse gene encoding dihydrofolate reductase (EC 1.5.1.3) that is oriented in the opposite direction to that of dihydrofolate reductase transcription. These opposite-strand RNAs are 180-240 nucleotides long, differing in length at their 5' ends. They are abundant in nuclear RNA and are not polyadenylylated. We suggest that the promoter region of the dihydrofolate reductase gene functions in a bidirectional manner to produce both these RNAs and dihydrofolate reductase mRNAs.

Animals↗
PubMed1985

Ultrastructural features of minute chromosomes in a methotrexate-resistant mouse 3T3 cell line.

The Miller spreading procedure was applied to mouse metaphase spreads of methotrexate-resistant 3T3 cells that contain large numbers of minute chromosomes and dihydrofolate reductase genes. There is substantial variation in both size and numbers of minutes in individual cells, the smallest of which (estimated as 5 X 10(3) kilobase pairs) would be undetected by standard light microscopic analyses. Minute chromosomes are composed of nucleosomal chromatin, which is organized into typical higher order fibers that are folded to form rosette-like structures characteristic of normal chromosome organization. There is no evidence that the DNA in minutes is linear. Minutes exist singly and in pairs, and members of a pair are connected by higher order chromatin fibers, suggesting that they are topologically interlocked. They are often closely apposed to chromosomal telomeres or arms, a configuration that may be involved in their distribution at mitosis. In addition to typical minutes, which do not possess kinetochores, a small marker chromosome possessing all of the features of a centromere region is present in parental and resistant cells. An unusual feature of this cell line is the retention of resistance, minute chromosomes, and amplified dihydrofolate reductase genes; most methotrexate-resistant mouse cell lines with minute chromosomes lose these properties when grown in the absence of methotrexate.

Animals↗
PubMed1982

Effects of NusA protein on transcription termination in the tryptophan operon of Escherichia coli.

Termination of transcription at the end of the tryptophan (trp) operon of E. coli at the trp t site is very efficient in vivo, but is only 25% efficient in vitro. To try to resolve this discrepancy, we have altered numerous parameters and report here on the modifications that bring the in vitro results into closer agreement with the in vivo ones. Lowering the concentration of UTP (but not ATP, CTP or GTP) in the transcription mix can greatly improve termination at trp t. With three other terminators structurally similar to trp t, there is no detectable effect of reducing the concentration of any of the four triphosphates. This response at trp t to low UTP is therefore both nucleotide-specific and terminator-specific, suggesting that apparently minor structural differences may still have profound effects upon termination. Increased specificity and sensitivity may also be provided by the NusA protein, which causes RNA polymerase to pause at trp t and at the 1:2 stem of the trp attenuator. NusA protein also enhances termination at trp t, an effect similar to the low UTP response. Further, termination can be slightly improved by including rho factor, resulting in an overall efficiency of almost 100% at trp t.

Bacterial Proteins↗
PubMed1982

Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of EScherichia coli.

We have devised a method to specifically incorporate deoxyribonucleotide base analogs in vitro into either strand of the tryptophan (trp) operon attenuator region, using primed synthesis on bacteriophage M13 derivatives carrying cloned trp attenuator DNA. We have employed these techniques to extend previous studies implicating both RNA-RNA and RNA-DNA interactions in transcription termination in an attempt to determine the nature of the contribution from the template DNA molecule in termination regions. In general, we find that the dramatic effects upon transcription termination seen with base analog incorporation into mRNA do not occur when similar analogs are incorporated into the DNA. Only the analog 2,6-diaminopurine deoxyribonucleotide triphosphate (dDapTP), which strengthens A.T or A.U base pairing, elicits a significant response: in the template DNA strand, the presence of this analog increases read-through at the trp attenuator. The analog 5-bromouracil deoxyribonucleoside triphosphate (BrdUTP), which also strengthens pairing with its complementary base, has no detectable effect on termination when it is placed in either strand of the trp attenuator or the mutant attenuator trp a1419. Surprisingly, though the analog 5-iodocytosine deoxyribonucleoside triphosphate (IdCTP) does not affect termination, it has a great effect on initiation of transcription, depressing trp promoter activity as well as stimulating transcription from other regions. These results support the postulated interaction between terminal uridines in mRNA and the template DNA strand in enhancing termination and suggest that there are no significant additional contributions from the DNA. In addition, the novel use of M13 derivatives for incorporating analogs into the DNA on a preparative scale provides a technique for introducing mutations in a general but controlled fashion as a new means for studying other regulatory regions.

DNA, Bacterial↗
PubMed1981

Rho-independent termination: dyad symmetry in DNA causes RNA polymerase to pause during transcription in vitro.

Termination of transcription by RNA polymerase at rho-independent sites appears to depend primarily upon two structural features, a region of GC-rich dyad symmetry in the DNA preceding the stop point and a stretch of uridines at the 3' end of the transcript. The possibility that the former might be responsible for slowing elongation prompted us to perform a kinetic analysis of transcription across the leader and terminator regions of the E. coli tryptophan (trp) operon. Regions where the elongation rate is dramatically slowed or stopped are identifiable because they generate discrete transcript hands on a gel. Species derived from pause sites, unlike those resulting from termination sites, are transient and detectable only within the first two minutes of transcription, since polymerase eventually resumes elongation. At two mutant trp attenuator sites (trp a135 and trp a1419), where termination is incomplete or absent in vitro, a substantial pause is nevertheless observed. Likewise, a significant pause occurs at trp t, the termination site at the end of the operon. Our experiments also reveal a major pause site at about position 90 in the trp leader sequence, just past a region of dyad symmetry. The RNA hairpin corresponding to this site is U-rich, and pausing is strongly enhanced by incorporation of BrUTP. In contrast, this analog does not affect pausing at the attenuator or terminator sites with hairpins that are GC-rich. These results strongly support the hypothesis that pausing of the polymerase is an obligatory prelude to rho-independent termination. Moreover, the termination event evidently results from consecutive but discrete responses to separate structural features of these sites.

Base Sequence↗
PubMed1981

Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro.

Termination of transcription by Escherichia coli RNA polymerase in vitro appears to depend primarily on two structural features of the termination site--a G+C-rich region of dyad symmetry and a series of terminal uridine residues in the transcript. To determine whether these two features are sufficient to specify rho-independent termination in vitro, we have introduced new sequences within a tryptophan (trp) operon structural gene to create two sites with these characteristics. Transcription with wild-type RNA polymerase in vitro demonstrates that discrete termination occurs at one of these new sites, although at a low level. Use of the mutant RNA polymerase rpo203, which is more sensitive to certain weak terminators than is the wild-type enzyme, increases termination at both sites. We have compared the activity of our synthetic terminators with those of several termination sites in the E. coli trp operon. Under normal conditions of transcription in vitro, termination becomes more efficient with an increase in the length of the stem in the RNA hairpin or an increase in the number of consecutive uridine residues. Transcription with the rpo203 polymerase and with ribonucleotide analogs gives changes consistent with these general trends. These results support a model for termination involving separate but essential roles for the RNA hairpin and the stretch of uridines in the transcript.

Base Sequence↗
PubMed1978

Effects of gentamicin on trypsin, chymotrypsin, and collagenase.

The effects of gentamicin on three proteolytic enzymes were studied. Gentamicin was tested at concentrations of 0.5-500 microgram/ml. Trypsin was tested at 0.5 microgram/ml using p-tosyl-L-arginine methyl ester and at 0.1 and 0.5 microgram/ml using azocoll as the substrate. Chymotrypsin was tested at 0.1 and 0.5 microgram/ml with azocoll. A soluble [14C]collagen assay was used to measure activity of collagenase derived from Clostridium histolyticum. The profiles of proteolytic activity vs. gentamicin concentration were similar for all three enzymes. At lower concentrations of gentamicin (less than 70 microgram/ml), there were two peaks of enhanced protease activity generally followed by inhibition. These unusual multiphasic effects of gentamicin on three different proteases are not presently understood, but they imply a previously unreported mode of action for this antibiotic.

Chymotrypsin↗