Biomedical subjects

J Lingner

Publications and source records attributed to J Lingner.

23 records · Page 2Linked to original sources

PubMed1994

Telomerase RNAs of different ciliates have a common secondary structure and a permuted template.

Telomerase is composed of protein and RNA. The RNA serves as a template for telomere DNA synthesis and may also be important for enzyme structure or catalysis. We have used the presence of conserved sequence elements in the promoter and template regions to amplify by PCR the telomerase RNA genes from six different hypotrichous ciliates: Oxytricha nova, Oxytricha trifallax, Stylonychia mytilis, Stylonychia lemnae, Euplotes aediculatus, and Euplotes eurystomus. RNaseH cleavage of the O. nova RNA in extracts by use of a complementary oligonucleotide leads to loss of telomerase activity, supporting the identification. Primary sequence and biochemical experiments suggest that the templates of Oxytricha and Stylonychia are circularly permuted relative to that of E. aediculatus. On the basis of the pause sites, the former two add G4T4 during a single primer elongation cycle, whereas E. aediculatus adds G3T4G. The only primary sequence element outside the template that is conserved between these phylogenetically distant telomerase RNAs is the sequence 5'-(C)UGUCA-3', which precedes the template regions by exactly two bases. We propose a common secondary structure model that is based on nucleotide covariations, a model which resembles that proposed previously for tetrahymenine telomerase RNAs.

Animals↗

PubMed1993

3'-end labeling of RNA with recombinant yeast poly(A) polymerase.

Two commonly used methods to end-label RNA-molecules are 5'-end labeling by polynucleotide kinase and 3'-end labeling with pCp and T4 RNA ligase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepin 5'-triphosphate (3'-deoxy-ATP) which is added by poly(A) polymerase. For a synthetic RNA it is shown that 40% of cordycepin becomes incorporated when the nucleotide is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at its 3'-end. The reaction is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNA-molecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.

Base Sequence↗

PubMed1991

Cloning and expression of the essential gene for poly(A) polymerase from S. cerevisiae.

Poly(A) polymerase is essential for the maturation of messenger RNA, adding tracts of adenosine residues to the 3' end of precursor RNA generated by endonucleolytic cleavage. This mechanism of mRNA 3' processing seems to be similar in yeast and in higher eucaryotes, although there are differences in the recognition signals in the pre-mRNA. Here we describe the cloning of the gene for yeast poly(A) polymerase. The enzyme is encoded by a single and essential gene located near the centromere on the left arm of chromosome 11. Poly(A) polymerase purified from recombinant Escherichia coli has the same physical and biochemical properties as the yeast enzyme. The yeast poly(A) polymerase shares features of sequence with its mammalian homologue.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of poly(A) polymerase from Saccharomyces cerevisiae.

Poly(A) polymerase was purified 22,000-fold to homogeneity from a whole cell extract of Saccharomyces cerevisiae with a yield of 22%. The enzyme is a monomeric polypeptide with a denatured molecular weight of 63,000. Incorporation of labeled ATP into acid-precipitable material by the purified enzyme proceeds faster with manganese than with magnesium ions. Various RNA homopolymers as well as Escherichia coli tRNA or rRNA can serve as primers. An RNA that terminates at the natural poly(A) site of the CYC1 gene is not more efficiently elongated than several nonspecific substrates, indicating the requirement for additional factors to provide specificity. Elongation of the primer is distributive. Covering of a poly(A) primer with poly(A)-binding protein reduces the enzyme's activity more than 10-fold.

Carrier Proteins↗

PubMed1989

Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes.

EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.

Amino Acid Sequence↗