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S Henikoff

Publications and source records attributed to S Henikoff.

At least 109 records · Page 6Linked to original sources
PubMed1992

Amino acid substitution matrices from protein blocks.

Methods for alignment of protein sequences typically measure similarity by using a substitution matrix with scores for all possible exchanges of one amino acid with another. The most widely used matrices are based on the Dayhoff model of evolutionary rates. Using a different approach, we have derived substitution matrices from about 2000 blocks of aligned sequence segments characterizing more than 500 groups of related proteins. This led to marked improvements in alignments and in searches using queries from each of the groups.

Algorithms↗
PubMed1992

A relationship between asparagine synthetase A and aspartyl tRNA synthetase.

A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase.

Amino Acid Sequence↗
PubMed1992

Unusual organizational features of the Drosophila Gart locus are not conserved within Diptera.

The Drosophila Gart locus consists of two genes. One gene encodes three enzymes in the de novo purine nucleotide biosynthesis pathway [glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART)]. The second gene lies within an intron of the purine gene and encodes a cuticle protein. To investigate the evolution of the Gart locus, the Chironomus tentans homolog was cloned by screening a genomic DNA library with a polymerase chain reaction product. This study shows that the interesting structural features of this locus conserved in two distant Drosophila species are not found in the Chironomus homolog. These features include the cuticle protein gene nested within an intron and the existence of an alternative transcript to yield a monofunctional enzyme. In addition, the extremely rapid divergence of coding sequence seen for members of the tandemly duplicated AIRS domain in Drosophila is found to be much less rapid in Chironomus.

Acyltransferases↗
PubMed1992

Position effect and related phenomena.

Position-effect variegation in Drosophila, the mosaic expression of genes juxtaposed to heterochromatin, remains an enigmatic long-range phenomenon. While the chromatin-conformation model has been challenged, compelling contrary evidence is lacking. Nevertheless, progress has been made in the genetic and molecular analysis of genes involved in the process of heterochromatin formation and in the characterization of genetic elements normally located in pericentric heterochromatin. In addition, telomeric position effect in yeast provides a new model system for the study of the quasi-stable inheritance of an inactivated state.

Animals↗
PubMed1992

PATMAT: a searching and extraction program for sequence, pattern and block queries and databases.

A program has been developed that provides molecular biologists with multiple tools for searching databases, yet uses a very simple interface. PATMAT can use protein or (translated) DNA sequences, patterns or blocks of aligned proteins as queries of databases consisting of amino acid or nucleotide sequences, patterns or blocks. The ability to search databases of blocks by 'on-the-fly' conversion to scoring matrices provides a new tool for detection and evaluation of distant relationships. PATMAT uses a pull-down, menu-driven interface to carry out its multiple searching, extraction and viewing functions. Each query or database type is recognized, reported, and the appropriate search carried out, with matches and alignments reported in windows as they occur. Any of the high scoring matches can be exported to a file, viewed and recalled as a query using only a few keystrokes or mouse selections. Searches of multiple database files are carried out by user selection within a window. PATMAT runs under DOS; the searching engine also runs under UNIX.

Amino Acid Sequence↗
PubMed1992

Somatic instability of a Drosophila chromosome.

A mitotically unstable chromosome, detectable because of mosaic expression of marker genes, was generated by X-ray mutagenesis in Drosophila. Nondisjunction of this chromosome is evident in mitotic chromosome preparations, and premature sister chromatid separation is frequent. The mosaic phenotype is modified by genetic elements that are thought to alter chromatin structure. We hypothesize that the mitotic defects result from a breakpoint deep in the pericentric heterochromatin, within or very near to the DNA sequences essential for centromere function. This unique chromosome may provide a tool for the genetic and molecular dissection of a higher eukaryotic centromere.

Animals↗
PubMed1992

Detection of Caenorhabditis transposon homologs in diverse organisms.

Although transposons that move via DNA intermediates are common in bacteria, invertebrates, and plants, none have been clearly documented in vertebrates and certain other classes of organisms. One such family of transposons includes invertebrate elements related to Caenorhabditis elegans Tc1. Blocks of aligned protein segments derived from this family were used to search a nucleotide sequence databank. Among the relatives detected were known bacterial insertion elements, revealing the ancient origin of the family. Furthermore, a Tc1-like homolog was detected in a catfish, raising the possibility that this valuable tool of C. elegans genetics can be used with vertebrate genomes. This study illustrates the use of multiple protein blocks for detection and evaluation of distant relationships.

Amino Acid Sequence↗
PubMed1991

Automated assembly of protein blocks for database searching.

A system is described for finding and assembling the most highly conserved regions of related proteins for database searching. First, an automated version of Smith's algorithm for finding motifs is used for sensitive detection of multiple local alignments. Next, the local alignments are converted to blocks and the best set of non-overlapping blocks is determined. When the automated system was applied successively to all 437 groups of related proteins in the PROSITE catalog, 1764 blocks resulted; these could be used for very sensitive searches of sequence databases. Each block was calibrated by searching the SWISS-PROT database to obtain a measure of the chance distribution of matches, and the calibrated blocks were concatenated into a database that could itself be searched. Examples are provided in which distant relationships are detected either using a set of blocks to search a sequence database or using sequences to search the database of blocks. The practical use of the blocks database is demonstrated by detecting previously unknown relationships between oxidoreductases and by evaluating a proposed relationship between HIV Vif protein and thiol proteases.

Algorithms↗
PubMed1991

So what's new?

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Publishing↗
PubMed1991

A pairing-sensitive element that mediates trans-inactivation is associated with the Drosophila brown gene.

Position-effect variegation in Drosophila is the mosaic expression of a gene juxtaposed to heterochromatin by chromosome rearrangement. The brown (bw+) gene is unusual in that variegating mutations are dominant, causing "trans-inactivation" of the homologous allele. We show that copies of bw+ transposed to ectopic sites are not trans-inactivated by rearrangements affecting the endogenous gene. However, when position-effect variegation is induced on an ectopic copy by chromosome rearrangement, the allele on its paired homolog is trans-inactivated, whereas other copies of bw+ are not. This confirms that trans-inactivation is "chromosome local" and maps the responsive element to the immediate vicinity of brown. Subsequent P-transposase-induced deletions within the ectopic copy in cis to the rearrangement breakpoint caused partial suppression of trans-inactivation. Surprisingly, the amount of suppression was correlated with deletion size, with some degree of trans-inactivation persisting even when the P[bw+] transposon was completely excised. The chromosome-local nature of the phenomenon and its extreme sensitivity to small disruptions of somatic pairing leads to a model in which a regulator of the brown gene is inactivated by direct contact with heterochromatic proteins.

Animals↗
PubMed1991

rbcR [correction of rcbR], a gene coding for a member of the LysR family of transcriptional regulators, is located upstream of the expressed set of ribulose 1,5-bisphosphate carboxylase/oxygenase genes in the photosynthetic bacterium Chromatium vinosum.

An open reading frame, rbcR, was identified 226 bp upstream of rbcAB, i.e., the ribulose 1,5-bisphosphate carboxylase genes expressed in the phototrophic purple bacterium Chromatium vinosum. Several features reveal that rbcR encodes a member of the LysR family of transcriptional regulators, in which an anomalous content of lysine and arginine residues (Lys/Arg anomaly) was found. The expression of rbcR in Escherichia coli as a protein fused to the N-terminal region of beta-galactosidase led to reduced expression of rbcAB. Thus, rbcR is likely to encode a trans-acting transcriptional regulator of rbcAB expression in C. vinosum.

Amino Acid Sequence↗
PubMed1991

Playing with blocks: some pitfalls of forcing multiple alignments.

Block alignments of multiple amino acid sequences are useful representations of regions thought to share common ancestry and function. Often the block alignments are motivated by the expectation that a protein of interest is similar in function to members of a family of proteins. However, when alignments are forced by using ad hoc methods, it is often difficult to decide whether the proposed relationship is valid. Visual examination can be deceptive, especially when alignments are not carried out in the context of controls subjected to similar procedures. Even computer-aided methods can be misleading when biases are introduced. To illustrate some of the problems that can arise, a few examples from the literature are analyzed. It is concluded that when standard methods fail to find an interesting block alignment unaided by human intervention, then the result should be regarded with caution.

Amino Acid Sequence↗
PubMed1990

Ordered deletions for DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates.

A simple method is described for generating nested deletions from any fixed point in a cloned inset. Starting with a single-stranded phagemid template, T4 DNA polymerase is used to extend an annealed primer. This leads to a fully double-stranded circular molecule with a nick or small gap just 5' to the primer. Exonuclease III initiates progressive digestion from the resulting 3' end. Removal of timed aliquots and digestion with a single-strand specific endonuclease leads to a series of linear nested fragments having a common end corresponding to the 5' end of the primer. These molecules are circularized and used to transform cells, providing large numbers of deletion clones with targeted breakpoints. The 6-step procedure involves successive additions to tubes, beginning with a single-stranded template and ending with transformation; no extractions, precipitations or centrifugations are needed. Results are comparable to those obtained with standard Exonuclease III-generated deletion protocols, but there is no requirement for restriction endonuclease digestion or for highly purified double-stranded DNA starting material. This procedure provides a strategy for obtaining nested deletions in either direction both for DNA sequencing and for functional analysis.

Animals↗
PubMed1990

Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin.

Contrary to the traditional view that microtubules pull chromosomes polewards during the anaphase stage of meiotic and mitotic cell divisions, new evidence suggests that the chromosome movements are driven by a motor located at the kinetochore. The process of chromosome segregation involves proper arrangement of kinetochores for spindle attachment, followed by spindle attachment and chromosome movement. Mechanisms in Drosophila for chromosome segregation in meiosis differ in males and females, implying the action of different gene products in the two sexes. A product encoded at the claret locus in Drosophila is required for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. Here we show that the predicted amino-acid sequence of this product is related to the heavy chain of kinesin. The conserved region corresponds to the kinesin motor domain and includes the ATP-binding site and a region that can bind microtubules. A second region contains a leucine repeat motif which may mediate protein-subunit interactions necessary for attachment of chromosomes to the spindle. The mutant phenotype of chromosome nondisjunction and loss, and its similarity to the kinesin ATP-binding domain, suggest that the product encoded at claret not only stabilizes chromosome attachments to the spindle, but may also be a motor that drives chromosome segregation in female meiosis.

Adenosine Triphosphatases↗
PubMed1990

Finding protein similarities with nucleotide sequence databases.

In this chapter we describe strategies for the searching of translated nucleotide sequence databases. By applying standard searching techniques developed for protein databases, we have found that previously unrecognized homologies can be detected. In addition, we have shown that extremely high sensitivity can be obtained using the scoring matrix strategy for short regions of similarity. The latter approach is particularly effective for detecting homologs found at the ends of sequences and within data of poor quality. These individual methods are demonstrated for the LysR family of bacterial activator proteins. Successive applications of these methods allow for sensitive detection of complex relationships, as demonstrated for the AraC family and for the complex LuxR-OmpR-NtrC families of bacterial activator proteins. Although our examples are drawn from bacterial sequences, these methods are likewise effective for higher eukaryotic genomic sequences, where protein-coding sequences are usually interrupted by introns. This should be particularly important in the future, since much of the expected increase in nucleotide sequence databases is likely to come from eukaryotic genomic sequencing projects.

Amino Acid Sequence↗
PubMed1990

Position-effect variegation after 60 years.

Position-effect variegation in Drosophila--the mosaic expression of a gene placed adjacent to a junction between euchromatin and heterochromatin--remains an enigma. However, new insights are being gained from recent studies of genetic modifiers, new model systems, and variegating genes showing exceptional behavior.

Animals↗