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R J Crouch

Publications and source records attributed to R J Crouch.

50 records · Page 3Linked to original sources
PubMed1983

A model for the involvement of the small nucleolar RNA (U3) in processing eukaryotic ribosomal RNA.

The nucleotide sequence of chick pre-rRNA between 5.8S and 28S rRNAs is 85% G + C and has the potential to form many different secondary structures. A model is presented in which a small nucleolar RNA, U3, and its associated proteins act as an RNA isomerase to position the pre-rRNA for processing. Cleavage could be performed either by a nuclease present in the U3RNP or by a ribonuclease directed to the proper form of the pre-rRNA.

Animals↗
PubMed1983

Low levels of RNase H activity in Escherichia coli FB2 rnh result from a single-base change in the structural gene of RNase H.

The DNA coding for RNase H from a mutant strain of Escherichia coli (FB2) was cloned into plasmid pBR322. DNA sequence analysis and the exchange of a portion of the mutant and wild-type genes revealed that a single-base alteration (C-->T) in the coding region of the structural gene for RNase H is responsible for the difference in RNase H activity of the wild-type and mutant cells.

Base Sequence↗
PubMed1981

Selective inhibition of RNase H by dextran.

Ordinarily, ribonuclease H hydrolyzes poly(rA) . poly(dT) and phiX174DNA-RNA at equal rates. Here we show that in the presence of dextran, the degradation of poly(rA) . poly(dT) is inhibited, while that of phi 174DNA-RNA is not. A similar inhibition by sucrose is found to be due to trace contamination of dextran in the sucrose. Ribose, deoxyribose, and a number of other saccharides fail to inhibit RNase H. In experiments where the two substrates are presented in the presence of the inhibitor, the kinetics indicates that both molecules are recognized by the enzyme, but only the phi X174DNA-RNA is degraded. That is, dextran does not interfere with the recognition site, but rather blocks hydrolysis. It is proposed that the ability of dextran to confer selectivity toward different substrates reveals a potential regulatory mechanism for RNase H activity which may represent a control step in the initiation of DNA synthesis.

Dextrans↗
PubMed1980

Isolation and mapping of a mutation in Escherichia coli with altered levels of ribonuclease H.

A mutant of Escherichia coli with altered levels of ribonuclease (RNase) H was isolated after mutagenesis with ethyl methane sulfonate. A procedure for assaying RNase H in partially purified extracts was used to screen approximately 1,500 colonies for variations in RNase H activity. Confirmation of a lower level of RNase H in the mutant was accomplished by analysis of RNase H in sodium dodecyl sulfate-polyacrylamide gels. By Hfr, F', and P1 transduction mapping, the genetic locus responsible for the lower levels of RNase H was located at 5.1 min on the E. coli chromosome. This mutation (rnh) represents a new locus on the E. coli chromosome. The only phenotypic characteristic of this mutation which has been observed to date is the lower level of RNase H (30% of parental values).

Chromosome Mapping↗
PubMed1979

Localisation of an endonuclease specific for double-stranded RNA within the nucleolus and its implication in processing ribosomal transcripts.

Nucleoli of both chick embryos and mouse Ehrlich ascites cells contain an enzymatic activity that is very similar to RNase DII, an enzyme isolated from total chick embryos for its ability to degrade double-stranded RNA. The enzyme can be extracted by low salt/EDTA from nucleoli and is associated with pre-ribosomal 80-S and 55-S particles. Under ionic conditions which are inhibitory for the nucleolytic activity the transcript in vitro of nucleoli is not processed and sediments around 45 S. Under salt conditions which are optimal for the nucleolar enzyme the nucleolar transcripts are cleaved to distinct intermediate-sized molecules. Addition of the chicken RNase DII or RNase III to the nucleolar transcription system results in a similar shift of the chain length of the RNA molecules. It is concluded that a nucleolar RNase recognizing double-stranded regions in the pre-ribosomal RNA is involved in the maturation of ribosomal RNA.

Animals↗
PubMed1977

Isolation and characterization of two enzymatic activities from chick embryos which degrade double-stranded RNA.

Enzymatic activities capable of degrading double-stranded RNA have been solubilized from whole 9-day-old chick embryos and separated by ion exchange chromatography on DEAE-cellulose into two classes, designated nucleases DI and DII. Nuclease DI exhibits an absolute requirement for Mn2+ in the range of 5 to 10 mM. Monovalent cations, including K+, Na+, and NH4+, are inhibitory. The molecular weight of DI is 60,000 to 62,500 as estimated from sedimentation in sucrose density gradients. Following gradient fractionation, nuclease DI possesses the ability to degrade several substrates exhibiting a 250-fold preference for poly(rC) as compared to poly(rC)-poly(rG). The activity responsible for degrading double-stranded RNA functions as an endonuclease generating oligonucleotides with 5'-phosphate termini. Nuclease DII requires both monovalent and divalent cations. Optimal degradation of poly[r(A-U)] is seen at 75 to 100 mM salt and 0.5 to 1.0 mM MgCl2 or MnCl2. The molecular weight estimated from sucrose gradient sedimentation is in the range of 38,000 to 40,000. Nuclease DII acts endonucleolytically producing oligonucleotides terminating in 5'-phosphates. During the isolation and characterization of nucleases DI and DII, a third activity was detected which degrades single-stranded RNA substrates but which, in the presence of either DII or RNase H, significantly enhances the degradation of poly[r(A-U)] or poly(rA)-poly(dT) substrates.

Animals↗
PubMed1975

Cleavage of adenovirus messenger RNA and of 28S and 18S ribosomal RNA by RNase III.

Escherichia coli ribonuclease III cleaves adenovirus messenger RNA and mammalian 28S and 18S ribosomal RNA. Fragmentation is not random, but in each case a specific collection of products is generated. This points to the potential use of the enzyme as a tool for specific fragmentation of RNA. Cleavage by RNase III abolishes the capability of adenovirus messenger RNA to direct cell-free synthesis of virus polypeptides.

Adenoviridae↗
PubMedDate not supplied

Genetic methods for the detection of microbial pathogens. Identification of enterotoxigenic Escherichia coli by DNA colony hybridization: collaborative study.

Enteropathogenic Escherichia coli strains may produce a cholera-like, heat-labile enterotoxin (LT) as a virulence factor. The gene that codes for LT can be purified by recombinant DNA techniques and used as a genetic probe for DNA hybridization. These probes detect enterotoxigenic strains as well as strains that may not manifest toxin production but carry the genetic information to do so. In this study, 13 laboratories tested 3 known and 25 unknown (10 positive and 15 negative) cultures of E. coli for the presence of the LT gene. The isolates had been tested and classified by the mouse Y-1 adrenal cell test and an enzyme-linked immunosorbent assay. Cultures were spotted on nitrocellulose filters on MacConkey agar and incubated. Colonies were lysed in situ and their DNA was hybridized to 32P-labeled, purified LT gene DNA (provided to the collaborators). Positive colonies were identified by autoradiography. Of 325 samples, 315 (96.9%) were identified correctly and 10 were misclassified; there were 6 false negative and 4 false positive identifications. Chi-square values indicated that the method agreed with the previous classification and was equally efficient in distinguishing positive and negative samples (95.7 and 98.1%, respectively). The method has been adopted official first action.

Autoradiography↗