scholarly journals The Chlamydomonas PF6 Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

2001 ◽  
Vol 12 (3) ◽  
pp. 739-751 ◽  
Author(s):  
Gerald Rupp ◽  
Eileen O'Toole ◽  
Mary E. Porter
Keyword(s):  
Insertional Mutagenesis ◽  
Expressed Sequence Tag ◽  
Human Testis ◽  
Flagellar Motility ◽  
Central Pair ◽  
Testis Cdna Library ◽  
Motility Mutant ◽  
Testis Cdna ◽  
Temporal And Spatial ◽  
Expressed Sequence

Efficient motility of the eukaryotic flagellum requires precise temporal and spatial control of its constituent dynein motors. The central pair and its associated structures have been implicated as important members of a signal transduction cascade that ultimately regulates dynein arm activity. To identify central pair components involved in this process, we characterized aChlamydomonas motility mutant (pf6-2) obtained by insertional mutagenesis. pf6-2 flagella twitch ineffectively and lack the 1a projection on the C1 microtubule of the central pair. Transformation with constructs containing a full-length, wild-type copy of the PF6 gene rescues the functional, structural, and biochemical defects associated with the pf6 mutation. Sequence analysis indicates that the PF6 gene encodes a large polypeptide that contains numerous alanine-rich, proline-rich, and basic domains and has limited homology to an expressed sequence tag derived from a human testis cDNA library. Biochemical analysis of an epitope-tagged PF6 construct demonstrates that the PF6 polypeptide is an axonemal component that cosediments at 12.6S with several other polypeptides. The PF6 protein appears to be an essential component required for assembly of some of these polypeptides into the C1-1a projection.

scholarly journals PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

1996 ◽  
Vol 132 (3) ◽  
pp. 359-370 ◽  
Author(s):  
E F Smith ◽  
P A Lefebvre
Keyword(s):  
Protein Interactions ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Central Pair ◽  
Central Apparatus ◽  
Immunofluorescence Labeling ◽  
Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

scholarly journals PF15p Is the Chlamydomonas Homologue of the Katanin p80 Subunit and Is Required for Assembly of Flagellar Central Microtubules

2004 ◽  
Vol 3 (4) ◽  
pp. 870-879 ◽  
Author(s):  
Erin E. Dymek ◽  
Paul A. Lefebvre ◽  
Elizabeth F. Smith
Keyword(s):  
Amino Acid ◽  
Significant Role ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Central Pair ◽  
Coding Sequence ◽  
Microtubule Severing ◽  
Central Apparatus

ABSTRACT Numerous studies have indicated that the central apparatus plays a significant role in regulating flagellar motility, yet little is known about how the central pair of microtubules or their associated projections assemble. Several Chlamydomonas mutants are defective in central apparatus assembly. For example, mutant pf15 cells have paralyzed flagella that completely lack the central pair of microtubules. We have cloned the wild-type PF15 gene and confirmed its identity by rescuing the motility and ultrastructural defects in two pf15 alleles, the original pf15a mutant and a mutant generated by insertional mutagenesis. Database searches using the 798-amino-acid polypeptide predicted from the complete coding sequence indicate that the PF15 gene encodes the Chlamydomonas homologue of the katanin p80 subunit. Katanin was originally identified as a heterodimeric protein with a microtubule-severing activity. These results reveal a novel role for the katanin p80 subunit in the assembly and/or stability of the central pair of flagellar microtubules.

Identification and characterization of a gene coding a novel isoform of DEAD-box protein

2002 ◽  
Vol 14 (3) ◽  
pp. 185 ◽  
Author(s):  
Lanlan Yin ◽  
JianMin Li ◽  
Hu Zhu ◽  
Min Lin ◽  
Lijun Cheng ◽  
...  
Keyword(s):  
Human Testis ◽  
Microarray Hybridization ◽  
Chain Reaction ◽  
Dead Box ◽  
Gene Coding ◽  
Testis Cdna Library ◽  
Testis Cdna ◽  
Polymerase Chain ◽  
Identification And Characterization

A gene coding a novel isoform of DEAD-box protein named testicular DEAD-box protein (tDbp), presumably involved in testicular function, was identified and characterized. Testicular DEAD-box protein was cloned from a human testis cDNA library. The cDNA microarray hybridization showed that it was expressed at a higher level in adult testis than in embryo testis. Reverse transcription-polymerase chain reaction indicated that tDbp was specifically expressed in testis, but not in some other tissues.

scholarly journals A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest–specific gene product

2003 ◽  
Vol 162 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Gerald Rupp ◽  
Mary E. Porter
Keyword(s):  
Gene Product ◽  
Insertional Mutagenesis ◽  
Growth Arrest ◽  
Coiled Coil ◽  
Specific Gene ◽  
Flagellar Motility ◽  
Wide Range ◽  
Motility Mutant ◽  
Regulatory Complex ◽  
Related Proteins

The dynein regulatory complex (DRC) is an important intermediate in the pathway that regulates flagellar motility. To identify subunits of the DRC, we characterized a Chlamydomonas motility mutant obtained by insertional mutagenesis. The pf2-4 mutant displays an altered waveform that results in slow swimming cells. EM analysis reveals defects in DRC structure that can be rescued by reintroduction of the wild-type PF2 gene. Immunolocalization studies show that the PF2 protein is distributed along the length of the axoneme, where it is part of a discrete complex of polypeptides. PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest–specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin. PF2 and its homologues appear to be universal components of motile axonemes that are required for DRC assembly and the regulation of flagellar motility. The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures.

A novel testis protein, RSB-66, interacting with INCA1 (inhibitor of Cdk interacting with cyclin A1)

2008 ◽  
Vol 86 (4) ◽  
pp. 345-351 ◽  
Author(s):  
Xu Chen ◽  
Tinghui Hu ◽  
Gang Liang ◽  
Maojun Yang ◽  
Shudong Zong ◽  
...  
Keyword(s):  
Round Spermatid ◽  
Human Testis ◽  
Amino Acid Residues ◽  
Yeast Two Hybrid ◽  
Cyclin A1 ◽  
Yeast Two Hybrid System ◽  
Testis Cdna Library ◽  
Testis Cdna ◽  
Α Helix ◽  
Two Hybrid

rsb-66 is a novel gene from a suppression subtracted hybridization (SSH) library of round spermatid-specific cDNAs against those of primary spermatocytes. It was found to be specifically expressed in round spermatids. To explore the function of RSB-66, a yeast two-hybrid system was used to screen for potential interacting partners in a human testis cDNA library. HSD45, also known as INCA1 (inhibitor of Cdk interacting with cyclin A1), was identified as one of the positive clones. The interaction between RSB-66 and INCA1 was demonstrated to occur by GST pull down and coimmunoprecipitation. Using immunofluorescence, RSB-66 was found to be specifically expressed in round spermatids, mainly in the cytoplasm. When being transfected into HeLa cells, RSB-66 and INCA1 were found to be co-localized principally in the cytoplasm. The α helix in the RSB-66 C terminal and two amino acid residues (tyr117 and his119) appear to be crucial for its function.

scholarly journals Molecular cloning and heterologous expression of an alternatively spliced human Mu class glutathione S-transferase transcript

1993 ◽  
Vol 294 (2) ◽  
pp. 373-380 ◽  
Author(s):  
V L Ross ◽  
P G Board
Keyword(s):  
Alternative Splicing ◽  
Evolutionary Process ◽  
Complete Sequence ◽  
Human Testis ◽  
Cdna Clones ◽  
Glutathione S Transferase ◽  
Testis Cdna Library ◽  
Alternatively Spliced ◽  
Testis Cdna ◽  
Illegitimate Transcription

Two cDNA clones encoding a new Mu class glutathione S-transferase (GST) have been isolated from a human testis cDNA library. Both clones are incomplete and appear to result from alternative splicing. One clone is missing the sequence encoding exon 4 and the other is missing exon 8. The complete sequence of the previously undescribed isoenzyme can be deduced from the two cDNA clones. This is the first report of alternative splicing in a GST transcript and may represent either a novel form of regulation in this multigene family or illegitimate transcription and experimental alternative splicing as part of the evolutionary process. By combining components from each clone a complete cDNA has been constructed and the encoded protein expressed in Escherichia coli. In general, the recombinant enzyme has relatively low activity when compared with all the previously described human Mu class GST isoenzymes.

Human testis cDNAs identified by sera from infertile patients: a molecular biological approach to immunocontraceptive development

1994 ◽  
Vol 6 (3) ◽  
pp. 297 ◽  
Author(s):  
ZG Liang ◽  
PA O'Hern ◽  
B Yavetz ◽  
H Yavetz ◽  
E Goldberg
Keyword(s):  
Human Testis ◽  
Vaccine Candidates ◽  
Contraceptive Vaccine ◽  
Immunological Infertility ◽  
Testis Cdna Library ◽  
Human Sera ◽  
Testis Cdna ◽  
Immunologic Infertility ◽  
Infertile Patients ◽  
Molecular Biological Approach

Sera from patients with known or suspected immunological infertility were used to screen a human testis cDNA library. A total of 59 sera detected 38 unique cDNA inserts of which four were testis specific by Northern blot analyses. One of these is a testis-specific isoform of calpastatin. Five additional clones, although not testis specific, were found to be testis abundant. The number and type of clones identified by these human sera suggests a possible aetiology for immunologic infertility. The testis-specific clones will be further characterized to establish their usefulness as contraceptive vaccine candidates.

Survey of a cDNA library from the turkey (Meleagris gallopavo)

Genome ◽  
2005 ◽  
Vol 48 (1) ◽  
pp. 12-17 ◽  
Author(s):  
L D Chaves ◽  
J A Rowe ◽  
K M Reed
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Expressed Sequence Tags ◽  
Expressed Sequence Tag ◽  
Meleagris Gallopavo ◽  
Whole Genome Sequence ◽  
Snp Discovery ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Important Species ◽  
Expressed Sequence

Genome characterization and analysis is an imperative step in identifying and selectively breeding for improved traits of agriculturally important species. Expressed sequence tags (ESTs) represent a transcribed portion of the genome and are an effective way to identify genes within a species. Downstream applications of EST projects include DNA microarray construction and interspecies comparisons. In this study, 694 ESTs were sequenced and analyzed from a library derived from a 24-day-old turkey embryo. The 437 unique sequences identified were divided into 76 assembled contigs and 361 singletons. The majority of significant comparative matches occurred between the turkey sequences and sequences reported from the chicken. Whole genome sequence from the chicken was used to identify potential exon–intron boundaries for selected turkey clones and intron-amplifying primers were developed for sequence analysis and single nucleotide polymorphism (SNP) discovery. Identified SNPs were genotyped for linkage analysis on two turkey reference populations. This study significantly increases the number of EST sequences available for the turkey.Key words: turkey, cDNA, expressed sequence tag, single nucleotide polymorphism.

Screening and analysis of differentially expressed genes from an alien addition line of wheat Thinopyrum intermedium induced by barley yellow dwarf virus infection

Genome ◽  
2004 ◽  
Vol 47 (6) ◽  
pp. 1114-1121 ◽  
Author(s):  
Shu-Mei Jiang ◽  
Long Zhang ◽  
Jun Hu ◽  
Rui Shi ◽  
Guang-He Zhou ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Expressed Sequence Tag ◽  
Barley Yellow Dwarf Virus ◽  
Differentially Expressed ◽  
Addition Line ◽  
Alien Chromosome ◽  
Thinopyrum Intermedium ◽  
Barley Yellow Dwarf ◽  
Yellow Dwarf Virus ◽  
Expressed Sequence

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.Key words: suppression subtractive hybridization (SSH), expressed sequence tag (EST), linker adaptor mediated polymerase chain reaction (LA-PCR), chromosome microdissection.

Characterization of expressed sequence tag-derived simple sequence repeat markers for 17 Linum species

Botany ◽  
2010 ◽  
Vol 88 (5) ◽  
pp. 537-543 ◽  
Author(s):  
Yong-Bi Fu ◽  
Gregory W. Peterson
Keyword(s):  
Simple Sequence Repeat ◽  
Expressed Sequence Tag ◽  
Sequence Repeat ◽  
Simple Sequence Repeat Markers ◽  
Linum Usitatissimum L ◽  
Evolutionary Studies ◽  
Expressed Sequence ◽  
Simple Sequence ◽  
Cultivated Flax

One major challenge in genetic and evolutionary studies of wild flax species is the lack of informative molecular markers. A set of 100 informative expressed sequence tag-derived simple sequence repeat (EST-SSR) primer pairs developed in cultivated flax ( Linum usitatissimum L.) were characterized on 35 Linum accessions representing 17 Linum species for their transferability to other Linum species. Ninety-nine primer pairs displayed scorable polymorphisms across 35 Linum samples and generated 627 bands likely from 121 loci. About 50% of the detected bands occurred only in three or fewer samples. A total of 393 bands, likely from 116 loci, were detected by 97 primer pairs in Linum bienne Mill. samples, but only up to 60 bands, likely from up to 39 loci, were revealed by 6 to 37 primer pairs in the samples of the other 15 Linum species. The L. bienne samples displayed 23.7% more EST-SSR variation than the L. usitatissimum samples. These characterized EST-SSR markers should be useful for future genetic diversity and evolutionary studies of Linum species, particularly for the progenitor of cultivated flax.

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