Biomedical subjects

A J Lustig

Publications and source records attributed to A J Lustig.

26 records · Page 2Linked to original sources

PubMed1992

Hoogsteen G-G base pairing is dispensable for telomere healing in yeast.

The G-rich strands of most eukaryotic telomeres are capable of forming highly folded structures in vitro, mediated, in part, through Hoogsteen G-G base pairing. The ability of most telomeres to form these structures has led to the suggestion that they play an important role in telomere addition. I have investigated this possibility in the yeast Saccharomyces cerevisiae through the use of an in vivo assay that measures healing via poly(G1-3T) addition onto plasmid substrates containing synthetic telomeres. Synthetic telomere healing is a highly size- and sequence-specific process that allows the discrimination of telomeres of differing efficiency. Plasmids containing synthetic telomeres with differing abilities to form secondary structures were tested in this assay for healing in vivo. The results of this study demonstrate that telomeres incapable of forming Hoogsteen base pairs nonetheless serve as efficient substrates for poly(G1-3T) addition, indicating that intramolecular Hoogsteen G-G base pairing is not essential for this process.

Base Composition↗

PubMed1992

C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.

Alleles↗

PubMed1990

Involvement of the silencer and UAS binding protein RAP1 in regulation of telomere length.

The yeast protein RAP1, initially described as a transcriptional regulator, binds in vitro to sequences found in a number of seemingly unrelated genomic loci. These include the silencers at the transcriptionally repressed mating-type genes, the promoters of many genes important for cell growth, and the poly[(cytosine)1-3 adenine] [poly(C1-3A)] repeats of telomeres. Because RAP1 binds in vitro to the poly(C1-3A) repeats of telomeres, it has been suggested that RAP1 may be involved in telomere function in vivo. In order to test this hypothesis, the telomere tract lengths of yeast strains that contained conditionally lethal (ts) rap1 mutations were analyzed. Several rap1ts alleles reduced telomere length in a temperature-dependent manner. In addition, plasmids that contain small, synthetic telomeres with intact or mutant RAP1 binding sites were tested for their ability to function as substrates for poly(C1-3A) addition in vivo. Mutations in the RAP1 binding sites reduced the efficiency of the addition reaction.

Base Sequence↗

PubMed1988

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.

Antibodies, Fungal↗

PubMed1987

Splicing of yeast nuclear pre-mRNA in vitro requires a functional 40S spliceosome and several extrinsic factors.

We have previously shown that extracts prepared from most of the yeast temperature-sensitive rna mutants are heat sensitive for pre-mRNA splicing in vitro, and that the products of the corresponding RNA genes are essential for the early stages of the splicing region. In this report, we demonstrate that most heat-inactivated mutant extracts do not form the spliceosome, suggesting that their gene products are likely to be involved in spliceosome formation. Heat-inactivated rna2 extracts, on the other hand, do form a splicing-dependent 40S complex containing uncleaved pre-mRNA exclusively. The pre-mRNA in the 40S complex can be converted to the splicing products in the presence of ATP and complementing extracts. These results demonstrate that: (1) the 40S complex formed in heat-inactivated rna2 extracts is a spliceosome (termed the rna2 delta spliceosome), (2) the spliceosome is a functional intermediate in the splicing pathway, and (3) the splicing process can be dissected into two steps, spliceosome formation and cleavage-ligation reactions. Additional results indicate that at least two extrinsic factors, as well as the RNA2 gene product, are required for complementation of the rna2 delta spliceosome. A three-step mechanism for nuclear pre-mRNA splicing in yeast is proposed.

Genes, Fungal↗

PubMed1986

The yeast RNA gene products are essential for mRNA splicing in vitro.

The yeast rna mutations (rna2-rna11) are a set of temperature-sensitive mutations that result in the accumulation of intron-containing mRNA precursors at the restrictive temperature. We have used the yeast in vitro splicing system to investigate the role of products of the RNA genes in mRNA splicing. We have tested the heat lability of the in vitro mRNA splicing reaction in extracts isolated from mutant and wild-type cells. Extracts isolated from seven of the nine rna mutants demonstrated heat lability in this assay, while most wild-type extracts were stable under the conditions utilized. We have also demonstrated that heat inactivation usually results in the specific loss of an exchangeable component by showing that most combinations of heat-inactivated extracts from different mutants complement one another. In three cases (rna2, rna5, and rna11), the linkage of the in vitro defect to the rna mutations was ascertained by a combination of reversion, tetrad, and in vitro complementation analyses. Furthermore, each heat-inactivated extract was capable of complementation by at least one fraction of the wild-type splicing system. Thus many of the RNA genes are likely to code for products directly involved in and essential for mRNA splicing.

Genes, Fungal↗

PubMed1986

Identification of yeast mutants with altered telomere structure.

The chromosomes of the yeast Saccharomyces cerevisiae terminate in a tract of simple-sequence DNA [poly(C1-3A)] that is several hundred base pairs long. We describe the identification of mutant yeast strains that have telomeric tracts that are shorter than normal. A genetic analysis of these strains indicates that these short telomeres are the result of single nuclear recessive mutations and that these mutations can be classified into two different complementation groups. The full expression of the mutant phenotype shows a very long lag (approximately equal to 150 cell divisions). From our analysis of these mutants as well as other data, we suggest that the duplication of the telomeric poly(C1-3A) tract involves two processes, semiconservative replication and untemplated terminal addition of nucleotides.

Base Sequence↗

PubMed1984

Long poly(A) tracts in the human genome are associated with the Alu family of repeated elements.

Long poly(dA).poly(dT) tracts (poly(A) tracts), regions of DNA containing at least 20 contiguous dA residues on one strand and dT residues on the complementary strand, are found in about 2 X 10(4) copies interspersed throughout the human genome. Using poly(dA).poly(dA) as a hybridization probe, we identified recombinant lambda phage that contained inserts of human DNA with poly(A) tracts. Three such tracts have been characterized by restriction mapping and sequence analysis. One major poly(A) tract is present within each insert and is composed of from 28 to 35 A residues. In each case, the poly(A) tract directly abuts the 3' end of the human Alu element, indicating that the major class of poly(A) tracts in the human genome is associated with this family of repeats. The poly(A) tracts are also adjacent to A-rich sequences and, in one case, to a polypurine tract, having the structure GA3-GA3-GA4-GA6-GA5-GA4. We suggest that repetitive cycles of unequal crossing over may give rise to both the long poly(A) and polypurine tracts observed in this study.

Bacteriophage lambda↗