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Biomedical subjects

S H Myster

Publications and source records attributed to S H Myster.

6 recordsLinked to original sources
PubMed1997

The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex.

Amino Acid Sequence↗
PubMed1996

The dynein gene family in Chlamydomonas reinhardtii.

To correlate dynein heavy chain (Dhc) genes with flagellar mutations and gain insight into the function of specific dynein isoforms, we placed eight members of the Dhc gene family on the genetic map of Chlamydomonas. Using a PCR-based strategy, we cloned 11 Dhc genes from Chlamydomonas. Comparisons with other Dhc genes indicate that two clones correspond to genes encoding the alpha and beta heavy chains of the outer dynein arm. Alignment of the predicted amino acid sequences spanning the nucleotide binding site indicates that the remaining nine clones can be subdivided into three groups that are likely to include representatives of the inner-arm Dhc isoforms. Gene-specific probes reveal that each clone represents a single-copy gene that is expressed as a transcript of the appropriate size (> 13 kb) sufficient to encode a high molecular weight Dhc polypeptide. The expression of all nine genes is upregulated in response to deflagellation, suggesting a role in axoneme assembly or motility. Restriction fragment length polymorphisms between divergent C. reinhardtii strains have been used to place each Dhc gene on the genetic map of Chlamydomonas. These studies lay the groundwork for correlating defects in different Dhc genes with specific flagellar mutations.

Amino Acid Sequence↗
PubMed1993

HLA class I sequence-based typing.

HLA oligogenotyping has been used successfully to characterize most phenotypically undetectable variants of class II genes. Limitations inherent to the class I system have, however, complicated the application of this and other molecular approaches to HLA class I typing. We have previously shown that HLA class II polymorphism can be analyzed by a SBT approach. Here we present a class I-SBT strategy that provides complete sequence information for the two most polymorphic exons of the HLA-A, -B, and -C alleles. HLA class I SBT is based on direct sequencing of PCR-amplified HLA-A, -B, and -C cDNAs and requires a total of six cDNA -PCR-sequencing reactions (two per locus) and 13 different oligonucleotides. Each combination of oligonucleotides per reaction results in locus-specific sequence ladders and allows identification of both alleles in heterozygotes. Application of HLA-A, HLA-B, and HLA-C SBT to 26 homozygous and 32 serologically heterozygous samples has resulted in the identification of 24 novel class I nucleotide sequences encoding 17 new major histocompatibility complex class I products. An unexpected high degree of heterogeneity was found at the HLA-C locus with 14 novel sequences. Although there was a good correlation between the serologic phenotypes and SBT results, HLA-C SBT of most HLA-C serologically homozygous samples (heterozygous for HLA-A and/or -B) revealed heterozygozity (six of eight). SBT, the first molecular typing approach that has been generalized to both class I and class II genes, may be of special interest in applications demanding high sensitivity and specificity, such as in paternity testing or in the evaluation of the effects of sequence allelism in the outcome of unrelated bone marrow transplantation.

Alleles↗
PubMed1992

Molecular cloning and sequence analysis of the cDNA for northern pike (Esox lucius) growth hormone.

The complete nucleotide sequence of the northern pike (Esox lucius) cDNA for pregrowth hormones was determined from clones derived from a pituitary gland cDNA library. Seventeen cDNA clones were isolated from a single mRNA species. A cDNA of 1,227 nucleotides was sequenced and found to encode a polypeptide of 209 amino acid residues, which included a putative signal sequence of 22 amino acid residues. Sequence comparison of the northern pike growth hormone gene to other known growth hormone genes revealed similarities closest to other members of the superorder Protacanthopterygii, which includes the Salmonidae family (i.e., salmon and trout).

Amino Acid Sequence↗
PubMed1989

Characterization and sequencing of the lac Y54-41 "uncoupled" mutant of the lactose permease.

The Escherichia coli strain carrying the lac Y54-41 gene encodes a mutant lactose permease which carries out normal downhill transport of galactosides but is defective in uphill accumulation. In this study, the mutant lac Y54-41 gene was cloned onto the multicopy vector pUR270. As expected, the cloned gene was shown to express normal downhill transport activity but was markedly defective in the uphill transport of methyl-beta-D-thiogalactopyranoside. Direct measurements of H+ transport revealed that the mutant permease can transport H+ with methyl-beta-D-thiogalactopyranoside but at a significantly reduced capacity compared to the wild-type strain. However, under conditions where the mutant and wild-type strains both transport lactose at similar rates, no detectable H+ transport was observed in the mutant strain. The entire cloned lac Y54-41 gene was subjected to DNA sequencing, and a single base substitution was found which replaces glycine 262 in the protein with a cysteine residue. Inhibition experiments showed that the mutant permease is dramatically more sensitive to three different sulfhydryl reagents: N-ethylmaleimide, p-hydroxymericuribenzoate, and p-hydroxymercuriphenylsulfonic acid. However, the lactose analogue, thiodigalactoside, was only marginally effective at protecting against inhibition in the mutant strain. The results are consistent with the idea that the sulfhydryl reagents are inhibiting the mutant permease activity by reacting with cysteine 262.

Base Sequence↗