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.

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 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.

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.

scholarly journals A gene that is related to SRY and is expressed in the testes encodes a leucine zipper-containing protein.

1995 ◽  
Vol 15 (7) ◽  
pp. 3759-3766 ◽  
Author(s):  
N Takamatsu ◽  
H Kanda ◽  
I Tsuchiya ◽  
S Yamada ◽  
M Ito ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Library ◽  
Cho Cells ◽  
Leucine Zipper ◽  
Leucine Zipper Motif ◽  
Hmg Box ◽  
Testis Cdna Library ◽  
Testis Cdna

SRY-related cDNA encoding a protein with a high-mobility-group (HMG) box and a leucine zipper motif, which was designated SOX-LZ, was isolated from a rainbow trout testis cDNA library. Comparison of this cDNA with the mouse homologous cDNA isolated from a testis cDNA library exhibits an overall amino acid sequence identity of 77%, which is in striking contrast to the abrupt loss of amino acid sequence homology outside the HMG box found among mammalian SRY genes. In both rainbow trout and mice, Northern (RNA) blot analyses have revealed the presence of a testis-specific 3-kb-long SOX-LZ mRNA, and this transcript appeared coincidentally with the protamine mRNA, suggesting its expression in the germ line. A recombinant HMG box region protein encoded by SOX-LZ could bind strongly with an oligonucleotide containing an AACAAT sequence, which is also recognized by mouse Sry and Sox-5. Upon cotransfection into CHO cells, SOX-LZ transactivated transcription through its binding motif when the region including the leucine zipper motif was deleted [SOX-LZ (D105-356)]; however, the intact SOX-LZ failed to transactivate. The intact SOX-LZ could form homodimers through the leucine zipper, which resulted in inhibition of DNA binding by the HMG box, while SOX-LZ (D105-356), which was incapable of dimerization, showed specific binding with the AACAAT sequence. Thus, the repressed transactivation of the intact SOX-LZ in CHO cells was primarily attributable to the low level of DNA binding of SOX-LZ homodimers.

scholarly journals Human and rat testis express two mRNA species encoding variants of NRD convertase, a metalloendopeptidase of the insulinase family

1997 ◽  
Vol 327 (3) ◽  
pp. 773-779 ◽  
Author(s):  
Véronique HOSPITAL ◽  
Annik PRAT ◽  
Catherine JOULIE ◽  
Dorra CHÉRIF ◽  
Robert DAY ◽  
...  
Keyword(s):  
Significant Degree ◽  
Human Testis ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Peptide Substrates ◽  
Rat Testis ◽  
Processing Enzymes ◽  
Nucleotide Insertion ◽  
Testis Cdna

Rat testis NRD convertase (EC 3.4.24.61) is a Zn2+-dependent endopeptidase that cleaves, in vitro, peptide substrates at the N-terminus of Arg residues in dibasic sites. This putative processing enzyme of the insulinase family of metallopeptidases exhibits a significant degree of similarity to insulinase and two yeast processing enzymes, Axl1 and Ste23. We report the cloning of two human testis cDNA species encoding isoforms of NRD convertase, hNRD1 and hNRD2. Whereas the hNRD1 transcript (3.7 kb) is equivalent to the previously characterized rat cDNA (rNRD1), hNRD2 and rNRD2 are 3.9 kb novel forms containing a nucleotide insertion encoding a 68-residue segment. This motif, which is inserted N-terminal of the Zn2+-binding site, HXXEH, is contained within the most conserved region among the insulinase family members. Analysis of the deduced primary sequences revealed 92% identity between rat and human orthologues. The human gene encoding NRD convertase was localized to chromosome 1p32.1-p32.2. Whereas NRD convertase is mostly expressed in testis and in 24 cell lines, low mRNA levels were detected in most of the 27 other tissues tested.

scholarly journals Expression of a novel reticulon-like gene in human testis

Reproduction ◽  
2002 ◽  
pp. 227-234 ◽  
Author(s):  
ZM Zhou ◽  
JH Sha ◽  
JM Li ◽  
M Lin ◽  
H Zhu ◽  
...  
Keyword(s):  
Short Form ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Human Testis ◽  
Nylon Membrane ◽  
Adult Testis ◽  
Fetal Testis ◽  
Pcr Products ◽  
Testis Cdna ◽  
Membrane Spanning

Identification of genes that are specifically expressed in the adult testis or the fetal testis is important for the study of genes related to the development of the testis. In this study, a human testis cDNA microarray was established. PCR products of 9216 clones from a human testis cDNA library were dotted on a nylon membrane; mRNA from adult and fetal testes were purified and probes were prepared by a reverse transcription reaction with testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes, and 96.8 and 95.4% of clones were positive, respectively. In total, 731 clones were differentially expressed: 592 were highly expressed in adult testis and 139 were highly expressed in fetal testis. Among these genes, a new reticulon (Rtn)-like gene was detected and named Rtn-T. Rtn-T was highly expressed in adult human testis. The cDNA of Rtn-T contains 3491 bp and the putative protein had 968 amino acids. This protein is homologous to the six known members of the Rtn family (KIAA0886, Rtn xL, reticulon 4a, Nogo-A, Nogo-A short form, and brain my043) but was different at the 5' end. All homologues originate from one gene, and result from both different promotor regions and different splicing. Rtn-T lacks the first exon and contains a second exon that is lacking in the other homologues. Rtn-T is shorter than KIAA0886, Rtn xL, reticulon 4a and Nogo-A, but longer than the Nogo-A short form and brain my043. Sequence analysis showed that Rtn-T protein has two hydrophobic regions that may be membrane-spanning domains. Expression profiles showed that Rtn-T is specifically and strongly expressed in testis. The results of the present study indicate that the Rtn-T gene is differentially expressed in adult and fetal testes and encodes a membrane protein that may have a function in testis development.

326. SPIF - A NOVEL TESTIS-SPECIFIC GENE AND ITS INTERACTION WITH PKA

2010 ◽  
Vol 22 (9) ◽  
pp. 126
Author(s):  
S. J. Tannock ◽  
E. A. McLaughlin ◽  
R. J. Aitken ◽  
S. D. Roman
Keyword(s):  
Northern Blotting ◽  
Full Length ◽  
Specific Gene ◽  
Binding Motif ◽  
Yeast Two Hybrid ◽  
Truncated Form ◽  
Binding Partners ◽  
Testis Cdna Library ◽  
Testis Cdna ◽  
Two Hybrid

The activation of protein kinase A (PKA) is strongly implicated in capacitation and sperm motility. However, the full pathway is yet to be elucidated. To identify potential PKA binding partners in sperm, a yeast two-hybrid assay was performed using the testis specific catalytic subunit (Cs) of PKA as the ‘bait’ to screen a mouse testis cDNA library. A novel cDNA clone termed Sperm PKA Interacting Factor (SPIF) was identified from the screen on three separate occasions. The interaction was confirmed by a protein pull-down using a C-terminal recombinant protein to SPIF and a PKACs antibody. During cloning and sequence analysis, SPIF was found to contain two isoforms; a full length (4770 bp) and a truncated form (2784 bp) with alternate start sites and an identical 3′ end, with only the full length isoform containing the PKA binding motif. SPIF was found to be testis specific using PCR and Northern Blotting with high expression levels in round spermatids and adult testis. The interaction between SPIF and PKA was further demonstrated with protein co-localisation in round spermatids and in the midpiece and flagellum of mouse sperm. In summary, we have identified a novel testis specific gene that in concert with PKA could prove to be an essential link in the incomplete capacitation pathway

Identification of a Novel Gene SRG4 Expressed at Specific Stages of Mouse Spermatogenesis

2004 ◽  
Vol 36 (5) ◽  
pp. 351-359 ◽  
Author(s):  
Xiao-Wei Xing ◽  
Lu-Yun Li ◽  
Gang Liu ◽  
Jun-Jiang Fu ◽  
Xiao-Jun Tan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Mouse Testis ◽  
Amino Acid Residues ◽  
Complex Process ◽  
Testis Cdna Library ◽  
New Gene ◽  
Testis Cdna ◽  
Gene 4

Abstract Spermatogenesis is a complex process. Two spermatocytes expression sequence tags (ESTs) BG101130 and BG100990 were found. Their putative amino acid sequences have high homology with rat Spag4 (sperm antigen 4). By electrical hybridization, a novel cDNA encoding polypeptide of 348 amino acid residues was identified from a mouse testis cDNA library. The new gene was designated as SRG4 (Spermatogenesis related gene 4) (GenBank accession No. AY307077). Results of Northern blot and RTPCR revealed that SRG4 expressed specifically in mouse testis. Changes of SRG4 expression in mouse different development stages were observed by RT-PCR. The SRG4 mRNA was hardly detected in 2 weeks postpartum, and expressed abundantly from 3 weeks later, reaching top lever at 4–5 weeks, while slightly down in aging mouse testis. Results of in situ hybridization showed that SRG4 gene expressed abundantly in spermatocytes, round spermatids. This indicated SRG4 gene may play an important role in mouse meiotic divisions of spermatocytes.

scholarly journals Identification of a Novel Leucine-rich Repeat Protein as a Component of Flagellar Radial Spoke in the Ascidian Ciona intestinalis

2003 ◽  
Vol 14 (2) ◽  
pp. 774-785 ◽  
Author(s):  
Potturi Padma ◽  
Yuhkoh Satouh ◽  
Ken-ichi Wakabayashi ◽  
Akiko Hozumi ◽  
Yuji Ushimaru ◽  
...  
Keyword(s):  
Polyclonal Antibody ◽  
Light Chain ◽  
Dynein Light Chain ◽  
Leucine Rich Repeat ◽  
Radial Spoke ◽  
Testis Cdna Library ◽  
Proteomics Approach ◽  
Testis Cdna ◽  
Lrr Protein ◽  
Leucine Rich Repeat Protein

Axonemes are highly organized microtubule-based structures conserved in many eukaryotes. In an attempt to study axonemes by a proteomics approach, we selectively cloned cDNAs of axonemal proteins by immunoscreening the testis cDNA library from the ascidianCiona intestinalis by using an antiserum against whole axonemes. We report here a 37-kDa protein of which cDNA occurred most frequently among total positive clones. This protein, named LRR37, belongs to the class of SDS22+ leucine-rich repeat (LRR) family. LRR37 is different from the LRR outer arm dynein light chain reported inChlamydomonas and sea urchin flagella, and thus represents a novel axonemal LRR protein. Immunoelectron microscopy by using a polyclonal antibody against LRR37 showed that it is localized on the tip of the radial spoke, most likely on the spoke head. The LRR37 protein in fact seems to form a complex together with radial spoke protein 3 in a KI extract of the axonemes. These results suggest that LRR37 is a component of the radial spoke head and is involved in the interaction with other radial spoke components or proteins in the central pair projection.

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