Biomedical subjects

S Henikoff

Publications and source records attributed to S Henikoff.

134 records · Page 8Linked to original sources

PubMed1983

Cloning exons of mapping of transcription: characterization of the Drosophila melanogaster alcohol dehydrogenase gene.

A novel method has been developed for determining the location of RNA termini and intron-exon boundaries using genomic clones. RNA is hybridized to a single-stranded M13 phage derivative of the genomic fragment of interest. S1 nuclease digestion results in an RNA-DNA hybrid corresponding to any transcript protected by the insert. Hydrolysis of the RNA with alkali, hybridization of the DNA with the opposite-strand M13 derivative of the genomic fragment and S1 nuclease digestion yields a mixture of pure exons. This mixture is analyzed by agarose gel electrophoresis and is cloned using excess blunt-ended M13 phage vector. Plaques that contain inserts are identified by transfer to nitrocellulose and hybridization to the genomic insert and are used without further purification. Cloning junctions are then determined by partial sequence analysis. These very nearly correspond to intron-exon boundaries or to either end of the transcript. When applied to the alcohol dehydrogenase gene from Drosophila, this method revealed clear differences between the 5' ends of embryo and adult transcripts both by blot hybridization and by analysis of 23 independent exon clones. In embryos, the mature transcript is apparently derived from three exons and in adults from four with the difference lying in the 5' untranslated portion of the transcript. The method should be particularly valuable for mapping and cloning transcripts that are rare or are not polyadenylated.

Alcohol Dehydrogenase↗

PubMed1983

Sequence of a Drosophila DNA segment that functions in Saccharomyces cerevisiae and its regulation by a yeast promoter.

We have determined the complete DNA sequence of a segment derived from Drosophila melanogaster that complements a yeast adenine-8 (ade8) mutation. This sequence, in conjunction with transcriptional analysis, predicts that a 206 amino acid sequence accounts for complementation. We have ligated a 1.5 kb promoter segment of the yeast alcohol dehydrogenase I (ADHI) gene just upstream of the open reading frame. This construction results in ADE8+ function that responds to glucose and ethanol in a manner similar to that for the chromosomal ADHI gene, as determined by a simple quantitative color assay for ADE8+ function.

Adenine↗

PubMed1983

A Drosophila metabolic gene transcript is alternatively processed.

We have determined the organization and transcription of a Drosophila DNA segment coding for the purine pathway enzyme, GAR transformylase. Because the transcript encoding this enzyme is very rare, we used a novel method for exon mapping without isolating a cDNA. Our results suggest a long polypeptide having multiple domains, with GAR transformylase at the COOH terminus preceded by an extensive repeat. Part of the same DNA segment specifies a shorter transcript, which consists of the same 5' exons but lacks the last three 3' exons. A polyadenylation signal within an intron allows this single gene to encode two polypeptides. We hypothesize that alternative processing of a transcript is a basis for channeling metabolic intermediates into two different pathways.

Acyltransferases↗

PubMed1983

Transcription terminates in yeast distal to a control sequence.

We have investigated transcription termination on a segment of Drosophila DNA that complements a yeast adenine-8 mutation. Poly(A)+ RNA transcribed from this segment in yeast terminates at multiple sites clustered just beyond an AAUAAA sequence implicated in polyadenylation of higher eucaryotic messages. Deletion analysis indicates that, in yeast, this sequence is not required for polyadenylation. Rather, transcription termination is signalled by a region that is upstream of the AAUAAA sequence. At least part of the control region appears to be an 8-base pair (bp) sequence also found in the termination control region of the yeast CYC1 gene. Termination sites for the various deletions show a clear sequence preference. These sites occur in clusters at least 50 bp downstream of the control region, suggesting similarities between termination in yeast and p-dependent termination in bacteria.

Animals↗

PubMed1981

Isolation of a gene from Drosophila by complementation in yeast.

Transformation of mutant yeast cells by cloned genomic DNA from a higher eukaryote has made it possible to isolate a Drosophila DNA sequence that complements a yeast adenine-8-mutation. A 0.8-kilobase poly(A)-containing RNA is transcribed from the cloned Drosophila segment in transformed yeast cells and can account for functional expression of the gene.

Adenine↗

PubMed1981

Position-effect variegation and chromosome structure of a heat shock puff in Drosophila.

Position-effect variegation was studied in Drosophila melanogaster using a rearrangement that places the 87C heat shock puff locus next to heterochromatin. After heat shock, the translocated 87C region failed to puff or to accumulate RNA in some nuclei, but puffed normally in other nuclei from the same individual. In situ hybridization experiments showed that the level of polyteny was not greatly affected at the inactive gene site. Therefore, the variable ability of a gene to be transcriptionally induced, rather than its dosage, is the basis of the position effect phenomenon. These experiments also showed that heat shock gene sequences are single-stranded in puffs under relatively gentle specimen preparation conditions, in contrast to the same sequences in unpuffed sites.

Animals↗

PubMed1977

Transcription at two heat shock loci in Drosophila.

Transcription at two heat shock loci in Drosophila melanogaster, in subdivisions 87A and 57C, was investigated by hybridization in situ with 3H-labeled messenger, nuclear RNA and whole cell RNA from cells cultured at elevated temperature. What appears to be the same 9 x 10(5) dalton heat shock message hybridizes at both sites. At 87a, little additional hybridization is obtained with nuclear or whole cell RNA. In contrast, at 87C the saturation level of hybridization by nuclear and whole cell RNA is much higher than that obtained with the message alone. This evidence for extensive hybridization at 87C but not at 87A by RNA distinct from the message is confirmed by the finding that excess nonradioactive message competes away most of the hybridization by 3H-labeled nuclear and whole cell RNA at the latter locus but not at the former. The noncompetable RNA migrates on an electrophoretic gel as a heterogeneous population of molecules, extending to sizes both larger and smaller than the message. These and other observations lead to the conclusion that a 87A transcription includes little more than sequences complementary to the 9 x 10(5) dalton message, while at 87C, there are sequences complementary to the same message and extensive additional sequences complementary to other species of RNA.

Base Sequence↗

PubMed1975

Localization of RNA from heat-induced polysomes at puff sites in Drosophila melanogaster.

Heat treatment of D. melanogaster tissue culture cells causes drastic changes in the pattern of protein synthesis and the size distribution of polysomes. Like the heat shock puffs on polytene chromosomes which appear while preexisting puffs regress, heat shock proteins appear on gels while the synthesis of preexisting proteins is sharply reduced, and heat-induced polysomes appear on gradients after preexisting polysomes have disappeared. Most of the poly(adenylic acid)-containing RNA isolated from high-temperature polysomes sediments in sucrose gradients and migrates in gels as a rather narrow band. This RNA is of sufficient size to code for one particular protein that is found to account for more than half of the total synthesis at high temperature. The RNA hybridizes in situ mainly at chromosome sub-division 87B, the site of the major heat shock puff.

Adenine Nucleotides↗