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S Henikoff

Publications and source records attributed to S Henikoff.

At least 127 records · Page 7Linked to original sources
PubMed1989

Trans-inactivation of the Drosophila brown gene: evidence for transcriptional repression and somatic pairing dependence.

Position-effect variegation in Drosophila is the variable inactivation of a gene that occurs when it is juxtaposed to heterochromatic regions of chromosomes. The brown gene, required for pteridine pigment in the eye, is unusual in that expression of the unrearranged homolog also is affected. This dominant effect can be very strong, as inactivation is detectable when as many as three trans copies of the gene are present. We show that pteridine reductions coincide with similar reductions in the accumulation of brown mRNA. The dominant effect is suppressed by certain altered structural configurations of the brown region, suggesting that somatic pairing is involved in the phenomenon. We propose that direct transmission of the altered chromatin structure characteristic of position-effect variegation (heterochromatinization) occurs between paired homologs in the region of the brown locus.

Animals↗
PubMed1989

A moveable 5' splice site in adenine phosphoribosyltransferase genes of Drosophila species.

In two distantly related Drosophila species, the use of alternate 5' splice sites to process an intron in pre-mRNA from homologous adenine phosphoribosyltransferase (APRT)-encoding genes led to RNAs encoding nonfunctional peptides in addition to APRT. The production of aberrantly spliced transcripts as a normal feature of gene expression supports a general model of eucaryotic gene evolution through alternative splicing and moveable splice junctions.

Adenine Phosphoribosyltransferase↗
PubMed1988

Detection of protein similarities using nucleotide sequence databases.

A simple procedure is described for finding similarities between proteins using nucleotide sequence databases. The approach is illustrated by several examples of previously unknown correspondences with important biological implications: Drosophila elongation factor Tu is shown to be encoded by two genes that are differently expressed during development; a cluster of three Drosophila genes likely encode maltases; a flesh-fly fat body protein resembles the hypothesized Drosophila alcohol dehydrogenase ancestral protein; an unknown protein encoded at the multifunctional E. coli hisT locus resembles aspartate beta-semialdehyde dehydrogenase; and the E. coli tyrR protein is related to nitrogen regulatory proteins. These and other matches were discovered using a personal computer of the type available in most laboratories collecting DNA sequence data. As relatively few sequences were sampled to find these matches, it is likely that much of the existing data has not been adequately examined.

Alcohol Dehydrogenase↗
PubMed1988

A large family of bacterial activator proteins.

At least nine different bacterial proteins belong to the LysR family. The gene sequence for one of these proteins is presented here. Six others (Escherichia coli LysR, IlvY, CysB; Salmonella typhimurium MetR; Rhizobium NodD; and Enterobacter cloacae AmpR) are known to activate other genes. Based on sequence alignments, each member of this family is predicted to have a helix-turn-helix DNA binding motif near its amino terminus. The combined evidence indicates that all nine proteins are related by common ancestry, are similarly folded, and are not detectably related to other known bacterial regulatory proteins. The DNA database searching procedure and other methods used in this study should be useful in detecting other groups of related proteins.

Bacterial Proteins↗
PubMed1988

The brown protein of Drosophila melanogaster is similar to the white protein and to components of active transport complexes.

The brown gene of Drosophila melanogaster is required for deposition of pteridine pigments in the compound eye and other tissues. We isolated a ca. 150-kilobase region including brown by microdissection and chromosome walking using cosmids. Among the cDNAs identified by hybridization to the cosmids, one class hybridized to a genomic region that is interrupted in two brown mutants, bw and In(2LR)CK, and to 2.8- and 3.0-kilobase poly(A)+ RNAs which are altered in the mutants. Nucleotide sequencing of these cDNAs revealed that the two transcripts differ as a consequence of alternative poly(A) addition and that both encode the same predicted protein of 675 amino acids. Searches of available databases for amino acid sequence similarities detected a striking overall similarity of this predicted protein to that of the D. melanogaster white gene. The N-terminal portion aligned with the HisP family of membrane-associated ATP-binding proteins, most of which are subunits of active transport complexes in bacteria, and to two regions of the multidrug resistance P-glycoprotein. The C-terminal portion showed a structural similarity to integral membrane components of the same complexes. Taken together with earlier biochemical evidence that brown and white gene products are necessary for uptake of a pteridine precursor and genetic evidence that brown and white proteins interact, our results are consistent with suggestions that these proteins are subunits of a pteridine precursor permease.

ATP-Binding Cassette Transporters↗
PubMed1987

Conserved arrangement of nested genes at the Drosophila Gart locus.

The Drosophila melanogaster Gart gene encodes three enzymatic activities in the pathway for purine de novo synthesis. Alternative processing of the primary transcript leads to the synthesis of two overlapping polypeptides. The coding sequence for both polypeptides is interrupted by an intron that contains a functional cuticle protein gene encoded on the opposite DNA strand. Here we show that this nested organization also exists at the homologous locus of a distantly related species, Drosophila pseudoobscura. In both species, the intronic cuticle gene is expressed in wandering larvae and in prepupae. Remarkably, there are 24 different highly conserved noncoding segments within the intron containing the cuticle gene. These are found upstream of the transcriptional start, at the 3' end, and even within the single intronic gene intron. Other introns in the purine gene, including the intron at which alternative processing occurs, show no such homologies. It seems likely that at least some of the conserved noncoding regions are involved in specifying the high level developmental expression of the cuticle gene. We discuss the possibility that shared cis-acting regulatory sites might enhance transcription of both genes and help explain their nested arrangement.

Amino Acid Sequence↗
PubMed1986

The Saccharomyces cerevisiae ADE5,7 protein is homologous to overlapping Drosophila melanogaster Gart polypeptides.

The Drosophila melanogaster Gart locus encodes two polypeptides specified by overlapping alternative transcripts. One transcript encodes only glycinamide ribotide synthetase (GARSase) on a 45,000 Mr polypeptide, while the other encodes GARSase aminoimidazole ribotide synthetase (AIRSase), and glycinamide ribotide transformylase (GARTase) on a 145,000 Mr polypeptide. In Saccharomyces cerevisiae, glycinamide ribotide synthetase and aminoimidazole ribotide synthetase are encoded at the ADE5,7 locus. Here I report the cloning, sequencing, and determination of the transcriptional organization of the yeast ADE5,7 gene. There is sufficient homology to align the predicted 802 amino acid ADE5,7 polypeptide with its Drosophila counterpart. These results, together with the sequence of the S. cerevisiae ADE8 gene encoding glycinamide ribotide transformylase, show that the entire Drosophila large polypeptide can be accounted for by the three enzymatic activities. A novel finding is that successive Drosophila domains are each homologous to the aminoimidazole ribotide synthetase portion of the yeast ADE5,7 gene, such that regions of homology with yeast aminoimidazole ribotide synthetase alternate from one of the Drosophila repeats to the other. Such a relationship suggests that the two Drosophila aminoimidazole ribotide synthetase domains together participate in catalysis. This model is consistent with a 20-year-old explanation of complex interallelic complementation such as that characterizing the gene segment encoding yeast aminoimidazole ribotide synthetase.

Acyltransferases↗
PubMed1986

Gene within a gene: nested Drosophila genes encode unrelated proteins on opposite DNA strands.

A pupal cuticle protein gene has been found within an intron of a Drosophila gene that encodes three purine pathway enzymatic activities. The intronic gene is encoded on the DNA strand opposite the purine pathway gene and is itself interrupted by an intron. Whereas the purine pathway gene is active throughout development, the intronic cuticle protein gene is expressed primarily over a 3 hr period in the abdominal epidermal cells of prepupae that secrete the pupal cuticle. Therefore, a housekeeping gene and a developmentally regulated gene function in a nested arrangement.

Acyltransferases↗
PubMed1986

Two Drosophila melanogaster mutations block successive steps of de novo purine synthesis.

Drosophila melanogaster purine auxotrophs ade2(1) and ade3(1) have been characterized biochemically. The ade2(1) strain is deficient in the fourth step of the de novo purine synthetic pathway catalyzed by phosphoribosylglycinamidine synthase (phosphoribosylformylglycinamide amidotransferase). The ade3(1) strain is deficient in the previous step catalyzed by phosphoribosylglycinamide formyltransferase (GART). The mutation responsible for the slightly leaky ade3(1) phenotype was characterized further. First, the mutant GART polypeptide was found to be of normal size and present at normal levels. Second, the GART-encoding region of the mutant was cloned, inserted into a yeast-Escherichia coli shuttle vector, and used to transform mutant yeast. Transformants showed very slight in vivo activity when compared to wild type, verifying that the mutation is in the GART coding sequence. Lastly, the region of the gene encoding GART activity from mutant and inbred parental strain flies was completely sequenced. A single base transition was found, leading to the substitution of a serine for a highly conserved glycine. These two mutations provide examples of blocks in the de novo purine synthetic pathway in a whole animal.

Acyltransferases↗
PubMed1986

Multiple purine pathway enzyme activities are encoded at a single genetic locus in Drosophila.

The Drosophila melanogaster Gart locus, known from previous work to encode the enzyme activity phosphoribosylglycinamide formyltransferase (GART), specifies two alternatively processed mRNAs and two proteins. We introduced the entire Gart locus into a Drosophila tissue culture cell line in which the locus is active. The resulting cell clones contained numerous copies of the locus and overproduced both mRNAs and both expected proteins, thus markedly facilitating analysis of these molecules. We assayed extracts of the clones for the activities of 10 enzymes important for de novo purine synthesis and found that, in addition to GART, two other purine pathway activities, phosphoribosylamine-glycine ligase (phosphoribosylglycinamide synthetase, GARS) and phosphoribosylformylglycinamidine cyclo-ligase (phosphoribosylaminoimidazole synthetase, AIRS), are similarly overproduced. All three activities are present together on the larger overproduced protein. A smaller protein appears to possess only GARS activity. Therefore, alternative mRNA processing can allow cells to produce enzyme activities in forms that are either linked or unlinked to other activities.

Acyltransferases↗
PubMed1985

A multifunctional protein possessing glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis.

Three activities on the pathway of purine biosynthesis de novo in chicken liver, namely, glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase, have been found to reside on the same polypeptide chain. Three diverse purification schemes, utilizing three different affinity resins, give rise to the same protein since the final material has identical specific activities for all three enzymatic reactions and a molecular weight on sodium dodecyl sulfate gels of about 110 000. A single antibody preparation precipitates all three activities and binds to the multifunctional protein obtained by two methods in Western blots. Partial chymotryptic digestion of the purified protein gives rise to two fragments, one possessing glycinamide ribonucleotide synthetase activity and the other containing glycinamide ribonucleotide transformylase activity.

Acyltransferases↗
PubMed1984

Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.

A method is described for the rapid generation and cloning of deletion derivatives well-suited for the sequencing of long stretches of DNA. This method is based on two useful features of exonuclease III: (1) processive digestion at a very uniform rate and (2) failure to initiate digestion at DNA ends with four-base 3'-protrusions. The method was applied to a 4570-bp Drosophila genomic DNA fragment cloned in the single-stranded phage vector M 13mp18. An ordered set of deletion clones was made by first cutting replicative form(RF) DNA with two restriction enzymes in the polylinker region of the vector between the Drosophila DNA and the sequencing primer binding site. One enzyme left a four-base 3'-protrusion that protected the remainder of the vector from exonuclease III attack, allowing unidirectional digestion of the insert sequence from the 5'-protruding end left by the other enzyme. Aliquots were removed at uniform intervals, treated with S1 nuclease, Klenow DNA polymerase, T4 DNA ligase, and then used to transfect competent cells. Most of the resulting clones derived from each aliquot were deleted to a predicted extent with only slight scatter, even for deletions of more than 4 kb. The method permits efficient isolation of clusters of deletion breakpoints within small preselected regions of large DNA segments, allowing nonrandom sequence analysis.

Bacteriophage lambda↗
PubMed1984

Sequences responsible for transcription termination on a gene segment in Saccharomyces cerevisiae.

We have mapped a signal sequence for mRNA 3'-end formation in Saccharomyces cerevisiae by using a Drosophila melanogaster DNA segment that complements a yeast adenine-8 mutation. That the 3' end of the transcript in S. cerevisiae nearly coincides with that in D. melanogaster is consistent with the possibility that mRNA termini are similarly determined in both organisms. Deletion analysis reveals that the complete signal is no more than 21 base pairs long. Part of the signal is the sequence TTTTTATA, which is seen in the termination region of several yeast genes. TTTTTATA appears to be able to act autonomously as a partial termination signal. The efficiency of the complete signal is affected by substitution of sequences downstream from it. This modulation of the effect of a signal is consistent with termination in S. cerevisiae, resembling rho-dependent termination in bacteria.

Animals↗