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Biomedical subjects

T Maniatis

Publications and source records attributed to T Maniatis.

211 records · Page 12Linked to original sources
PubMed1975

Nucleotide sequence of the rightward operator of phage lambda.

The sequence of 72 base pairs of the rightward operator (O-R) of bacteriophage lambda is presented as determined with simple and rapid methods for direct DNA sequencing. The sequence of an operator mutant is also described. The methods are of general use in sequencing DNA fragments with unique 5' ends up to 50 base pairs in length. Previous experiments have shown that this operator contains multiple sites recognized by the lambda phage repressor. We believe we have identified three of these sites.

Base Sequence↗
PubMed1973

Multiple repressor binding at the operators in bacteriophage lambda.

Short DNA duplexes are protected when lambda DNA is digested with nuclease in the presence of lambda repressor. As the ratio of repressor to operator is increased, six successively larger fragments are recovered, ranging in size from 35 to 100 base pairs, each of which binds repressor. Study of these fragments indicates that, at each of the two lambda operators (o(L) and o(R)), repressor first binds to a unique site (not necessarily terminal), and that five additional sites are then filled in linear right-ward or left-ward order. The nucleotide sequences and affinities for repressor of o(L) and o(R) are not identical, although six fragments of similar size are protected at each operator. Evidence is presented arguing against the existence of hairpin-like structures in the operator fragments, and, moreover, it is shown that the operator duplex does not unwind when repressor binds to it.

Autoradiography↗
PubMedDate not supplied

Detection of single base substitutions in total genomic DNA.

Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.

Base Sequence↗
PubMedDate not supplied

Simian virus 40 enhancer increases number of RNA polymerase II molecules on linked DNA.

Since the discovery of transcription enhancer sequences in the genomes of the DNA viruses simian virus 40(SV40) and polyoma virus, these elements have been shown to play an important part in the control of both viral and cellular gene expression. Enhancer elements act in cis to increase the amount of RNA produced from linked genes in a manner largely independent of distance and orientation. The mechanisms by which enhancers act are not understood; in particular, it is not known whether the enhancer-dependent increase in the level of stable RNA reflects an increase in the rate of transcription. To address this question, we have used an in vitro nuclear transcription assay to examine the effect of the SV40 enhancer on transcription of cloned human beta-globin genes transiently introduced into HeLa cells. We show here that the SV40 enhancer acts at least in part to increase the number of RNA polymerase II molecules transcribing the linked gene.

Cell Nucleus↗
PubMedDate not supplied

The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing.

A small set of distinctive short RNA molecules are found in the nuclei of all higher eukaryotic cells and yeast, in protein complexes known as 'small nuclear ribonucleoprotein particles', or snRNPs. Recent work has confirmed early suggestions that these particles form part of the machinery by which primary RNA transcripts are processed to their mature, functional form. In particular, snRNPs have been shown to be an integral part of the 'spliceosome', a multi-component complex involved in the removal of intron sequences from the coding regions of messenger RNA precursors.

Animals↗