PubMed Health⌕ Search

Biomedical subjects

G Krupp

Publications and source records attributed to G Krupp.

15 recordsLinked to original sources
PubMed1992

Initiator oligonucleotides for the combination of chemical and enzymatic RNA synthesis.

Transcription reactions with T7 RNA polymerase were performed in the presence of short oligonucleotides (oligos) with guanosine at the 3'-end. We obtained transcripts which had included these 'initiator oligos' at their 5'-termini. The oligos could contain mixtures of deoxyribo-, ribo-, 2'-O-methylated and biotinylated nucleotides. Only the 3'-terminal guanosine of these oligos was encoded in the template DNA at the transcription start point, in contrast to the remainder of the sequence. This 5'-terminal sequence is variable and eliminates the limitation that transcripts must start with a 5'-terminal guanosine. With a 5'-biotinylated dinucleotide, we obtained end-labeled RNAs suitable for nonradioactive RNA sequencing.

Biotin↗
PubMed1992

The acceptor stem in pre-tRNAs determines the cleavage specificity of RNase P.

As the result of an unusual RNase P specificity, some special, mature tRNAs have acceptor stems with eight instead of the common seven base pairs. The data from numerous studies suggest that some features in the tRNA domain of pre-tRNAs are important for this behaviour. Here, we show that only five base pairs in the acceptor stem of bacterial histidine tRNAs are required to obtain the changed cleavage site in an unrelated eukaryotic serine tRNA.

Base Composition↗
PubMed1991

Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P.

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.

Base Sequence↗
PubMed1991

Direct sequencing of double-stranded polymerase chain reaction-amplified 16S rDNA.

A number of different procedures have been developed for direct sequence analysis of PCR products. These methods rely on the cumbersome isolation of specific PCR products from agarose gels or the production of single-stranded template DNAs. In the approach presented here, we describe primers for the amplification of 16-S rDNA and a simple preparation of PCR product for sequencing.

Base Sequence↗
PubMed1990

The methylation of one specific guanosine in a pre-tRNA prevents cleavage by RNase P and by the catalytic M1 RNA.

Several modified nucleosides were introduced during in vitro RNA synthesis into a pre-tRNA(Ser). The pre-tRNAs were used as substrates for RNase P enzymes. No effects were observed with biotin-8-ATP or [alpha-S]-GPT, whereas with m7GTP, the cleavage reaction was completely inhibited. Analysis of pre-tRNAs which contained m7G at various positions has revealed a single base at the 5'-end of the acceptor stem where this modification absolutely prevents cleavage by catalytic M1 RNA, eukaryotic and prokaryotic RNase P holoenzymes. These results suggest that a critical contact must be made between pre-tRNA substrate and enzyme/ribozyme or that the approach of the potential cleaving agent (a positive magnesium ion) is made impossible by the positive charge at N-7 of the guanosine. In addition, we have shown that a pre-tRNA containing only m7G's can still form a complex with M1 RNA in a gel retardation assay.

Base Sequence↗
PubMed1990

Substrate recognition by RNase P and by the catalytic M1 RNA: identification of possible contact points in pre-tRNAs.

Modified bases were introduced into pre-tRNAs during in vitro RNA synthesis or by chemical modification. These RNAs were used as substrates for the catalytic M1 RNA and the RNase P holoenzyme from Schizosaccharomyces pombe. The synthetic approach permitted the insertion of 100% m7GTP into pre-tRNAs and this resulted in complete inhibition of the specific 5' processing reactions. Partially modified RNAs were obtained by chemical modifications of purines and uridines in the pre-tRNAs. This allowed detailed analyses of specific bases excluded in the products. With pre-tRNA(Ser) and initiator pre-tRNA(Met), strong effects were observed in the T arm and weaker effects in the anticodon stem. Only minor base exclusions were detected in the acceptor stem of pre-tRNA(Ser) and in the D arm of pre-tRNA(Met).

Base Sequence↗
PubMed1989

Unusual promoter-independent transcription reactions with bacteriophage RNA polymerases.

Efficient transcription reactions of DNA-dependent RNA polymerases require the presence of a specific promoter sequence. This report shows that in the absence of their cognate promoter, two bacteriophage RNA polymerases are capable of performing unusual transcription reactions: (i) the DNA template serves also as a primer for RNA synthesis and this leads to hybrid DNA/RNA molecules, (ii) if the DNA template forms a hairpin structure, the linear DNA can be transcribed via the 'rolling circle' mechanism.

Biological Evolution↗
PubMed1989

[Immunogenetics of mesangial IgA glomerulonephritis and Schönlein- Henoch purpura].

We have recently examined polymorphisms of immunoglobuline heavy chain genes at S mu and S alpha 1 switch region loci by RFLP technique and described differences of genotype frequencies between healthy controls and patients with IgA-nephritis and Henoch-Schönlein-Purpura respectively. In the present study we further characterized the heavy chain constant region of IgG2 and IgG3 and, in addition, a hypervariable region (D 14 S 1) of unknown function localized downstream. In the present larger patient cohort we confirmed a higher frequency of S alpha 1 7.4 kb homozygotes in patients with IgA-nephritis (but not SHP), i.e. 62.0% vs 38.8% in controls, p less than 0.001. Compared to controls RFLP-frequencies of the constant region of IgG2 were also different in patients with IgA-nephritis, but not RFLP frequencies of D 14 S 1 or of the constant region of IgG3. Patients with IgA-nephritis homozygote for 7.4 kb in S alpha 1 had more adverse renal outcome and hypertension and more severe histological lesions (i.e. interstitial fibrosis, p less than 0.005).

Gene Frequency↗
PubMed1988

RNA synthesis: strategies for the use of bacteriophage RNA polymerases.

This communication presents an overview of the methods for the synthesis of RNA with virtually any desired sequence. The use of transcription vectors is a powerful and convenient approach, if the cloned gene of interest has restriction sites at the proper positions. To overcome these limitations, two methods were developed where chemically synthesized oligodeoxynucleotides (oligos) were applied to define the 3' and 5' termini of the chosen transcripts. Both approaches use cloned genes and the template DNA is synthesized with DNA polymerase I (Klenow fragment). Consequently, there are no size limitations for the synthesized RNAs. For short transcripts, the entire template DNA (including the promoter sequence) can be synthesized chemically and any desired RNA sequence is possible. Recently, it was shown that even oligos without any promoter sequence can be used as template DNA for RNA polymerases. Experimental data are presented for two approaches. The first example is the synthesis of template DNA for T7 RNA polymerase where two oligos (initiator and terminator) define the beginning and end of transcripts from a cloned gene. The second example is the use of simple oligos as templates for RNA polymerases. The major problem encountered was the inaccurate transcription termination, which resulted in one or two additional nucleotides beyond the encoded sequence.

Base Sequence↗
PubMed1987

The RNA components of Schizosaccharomyces pombe RNase P are essential for cell viability.

The fission yeast Schizosaccharomyces pombe contains in the haploid genome one copy of the gene (designated rrkl) for the RNA components of RNase P. Gene disruption in diploid cells of one copy of rrkl resulted in a moderate reduction of the level of cellular RNase P activity. Haploidization by meiosis demonstrated that rrkl is required for cell growth. Thus, the RNA components of S. pombe RNase P are essential in vivo. This is similar to the situation in Escherichia coli.

Cell Division↗
PubMed1979

Rapid RNA sequencing: nucleases from Staphylococcus aureus and Neurospora crassa discriminate between uridine and cytidine.

Using end-labelled RNA, significant changes in base specificity of three nucleases have been detected under defined conditions. Staphylococcus aureus nuclease at pH 3.5 without Ca++ cleaves all Pyr-N bonds more uniformly and efficiently than RNase A, without any preference for Pyr-A bonds. At pH 7.5 in 10 mM Ca++ this enzyme cleaves all N-C and N-G bonds slowly, whereas N-U and N-A bonds are hydrolyzed rapidly. Hence, the base at the 3'- or at the 5'-side of a phosphodiester bond can determine the base specificity of S. aureus nuclease. - In absence of urea, Neurospora crassa endonuclease cleaves all phosphodiester bonds, but leaves all C-N bonds intact in 7 M urea. - RNase U2 at pH 3.5 cleaves A-N bonds more efficiently than at pH 5.0.

Base Sequence↗