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J S Lindsey

Publications and source records attributed to J S Lindsey.

10 recordsLinked to original sources
PubMed1997

A processed homeobox gene expressed in a stage-, tissue- and region-specific manner in epididymis.

Processed genes are generated by reverse transcription of mRNA and integration at a novel site in the genome and are typically transcriptionally silent. Here, we report that a processed gene on rat chromosome 4 that is highly related to the X chromosome-encoded rat Pem (r.Pem) homeobox gene is transcriptionally active. This processed gene, r.Pem2, is expressed at high levels in epididymis but not in any other tissues that express the r.Pem gene, including testis. Although r.Pem2, is expressed at high levels in epididymis but not in any other tissues that express the r.Pem gene, including testis. Although r.Pem2 lacks all the introns present in the r.Pem gene, it contains splice donor and acceptor sequences within the coding region, permitting it to be spliced and to potentially encode a 57 amino acid polypeptide. r.Pem2 transcripts accumulate in the caput and cauda regions of the epididymis but not in the initial segment. The r.Pem2 gene exhibits different regulation from the r.Pem gene; its expression is induced later during prepubertal development (between days 23 and 30 post partum) and independently of the presence of testosterone. Although the functional significance of r.Pem2 is unknown, its developmental regulation and its apparent acquisition of splicing sites during evolution are unique.

Amino Acid Sequence↗
PubMed1996

Pem: a testosterone- and LH-regulated homeobox gene expressed in mouse Sertoli cells and epididymis.

Few transcription factors in somatic cells of the testis and epididymis that could potentially regulate androgen-dependent developmental events during male gametogenesis have been identified. In this study we examined the regulation and expression of an orphan homeobox gene, Pem, which encodes a homeodomain related to those in the Prd/Pax gene family. RNase protection, in situ hybridization, and Northern blot analyses of wild-type and germ-cell-deficient mutant mice (W(V)/ W(V)) localized Pem transcripts to Sertoli cells of the testis. During prepubertal testicular development, Pem expression was dramatically induced on Day 9, approximately when germ cells are known to enter meiotic prophase. In adult mice, Pem transcripts were preferentially expressed in stages VII-VIII seminiferous epithelium, the androgen-dependent stages during which germ cells undergo the first step of meiosis. Pem gene expression depended on androgens and gonadotrophins, as demonstrated by a lack of expression in hypophysectomized mice, gonadotrophin-deficient hypogonadal (hpg) mutant mice, and androgen receptor-deficient (tfm) mutant mice. Injection of either testosterone or luteinizing hormone (LH) into hypophysectomized and hpg/hpg mice restored Pem expression in the testes to normal levels. The Pem gene was also shown to be specifically expressed in the proximal cauda and distal corpus regions of the epididymis, the regions where spermatozoa gain forward motility and fertilization competence. Pem expression in the epididymis did not depend on spermatozoa in the lumen of the testis, as shown in quaking (qk/qk) mutant mice, however, unlike in the testes, epididymal Pem expression required germ-cell-induced factors. Our results show that discrete cell types in male reproductive tissues transcribe and independently regulate the Pem homeobox gene. To our knowledge no transcription factors have previously been shown to depend on testosterone or LH for expression in Sertoli cells in vivo. Collectively, the data implicate Pem as a candidate to regulate a subset of androgen-dependent genes in the male reproductive system.

Animals↗
PubMed1996

The Pem homeobox gene. Androgen-dependent and -independent promoters and tissue-specific alternative RNA splicing.

The Pem gene encodes an atypical homeodomain protein, distantly related to Prd/Pax family members, that we demonstrate is regulated in a complex transcriptional and post-transcriptional manner. We show that the rat Pem genomic structure includes three 5'-untranslated (5'-UT) exons and four coding exons, three of which encode the homeodomain. Several alternatively spliced transcripts were identified, including one that skips an internal coding exon, enabling this mRNA to express a novel form of the Pem protein. Other alternatively spliced mRNAs were characterized that possess different 5'-UT regions, including a muscle-specific transcript. The different 5'-UT termini present in Pem transcripts conferred different levels of translatability in vitro. Two promoters containing multiple transcription initiation sites were identified: a distal promoter (Pd) in the first 5'-UT exon and a proximal promoter (Pp) located in the "intron" upstream of the first coding exon. The Pd was active in placenta, ovary, tumor cell lines, and to a lesser extent in skeletal muscle. In contrast, transcripts from the Pp were only detectable in testis and epididymis and were only expressed in epididymis in the presence of testosterone. To our knowledge no transcription factors have previously been identified that exhibit androgen-dependent expression in the epididymis.

Alternative Splicing↗
PubMed1996

The Pem homeobox gene: rapid evolution of the homeodomain, X chromosomal localization, and expression in reproductive tissue.

A hallmark of homeobox genes is their high degree of sequence conservation in distantly related species. Here, we report the chromosomal localization, sequence, and expression pattern of an orphan homeobox gene, Pem, that encodes a homeodomain (HD) that has undergone a surprisingly high rate of evolutionary change. The N-terminal portion of the Pem HD, which includes the first two alpha-helices, exhibits only 44% sequence identity between rat Pem (r.Pem) and mouse Pem (m.Pem). This N-terminal subdomain exhibited an extremely high frequency of nonsynonymous substitutions, severalfold higher than other regions of the Pem protein. In contrast, the third helix, which is known to confer most of the base-specific contacts of HDs with DNA, was almost identical in r. Pem and m.Pem. Several lines of evidence suggested that the rat and mouse genes that we identified as Pem genes are true homologues: (1) the r.Pem and m.Pem genes both reside on the X chromosome; (2) they possess identical exon/intron splice junctions; (3) they both encode a distinctive motif upstream of the HD that is unique to Pem; and (4) the only m.Pem-like gene we were able to identify in the rat genome other than r.Pem was a pseudogene, r.Pem-ps, whose sequence and chromosomal localization indicated that it was derived by reverse transcription and reinsertion into the genome. The functional r.Pem gene is selectively expressed in placenta, testis, epididymis, and ovary. This expression pattern is of interest since other genes transcribed in reproductive tissue have also been shown to undergo high rates of sequence divergence. The high rate of amino acid substitutions in the N-terminal region of the Pem HD suggests the possibility of species-specific directional selection.

Alternative Splicing↗
PubMed1996

An androgen-regulated homeobox gene expressed in rat testis and epididymis.

Homeobox genes encode DNA-binding proteins that regulate the transcription of subordinate downstream genes. In this study, we show that the Pem homeobox gene is expressed and regulated in a unique manner in neonatal and adult rats. Pem gene expression was primarily confined to reproductive tissue: epididymis, testis, ovary, and placenta. In the epididymis, Pem transcripts were localized by in situ hybridization analysis to the proximal cauda region, a site where spermatozoa gain fertilization competence. Pem mRNA levels dramatically increased between Days 21 and 26 postpartum in the epididymis, coincident with the induction of genes known to be responsive to testosterone (T), but in contrast to that of other genes examined, including the Hoxc-8 homeobox gene. Pem expression was shown to be T-dependent on the basis of an absence of Pem transcripts in the epididymides of hypophysectomized rats and restoration of normal Pem mRNA levels after administration of T. In the testis, Pem mRNA levels were elevated earlier (between Days 12 and 15 postpartum) and less dramatically than in epididymis. Pem gene expression in the testis was depressed after hypophysectomy, but normal levels of Pem expression were not restored by T treatment under the same conditions that permitted normal Pem expression in the epididymis. To our knowledge Pem is the first reported putative transcription factor that has been demonstrated to depend on androgens for expression in the epididymis, and thus Pem is a candidate as a regulator of androgen-dependent events in this tissue.

Androgens↗
PubMed1995

Flexible protocols improve parallel experimentation throughput.

Advanced chemical workstations offer the potential to substantially improve the productivity of experimental research. To fully exploit such technologies, effective scheduling of experiments is crucial. Chemists tend to define experimental protocols with rigid time constraints, although often the scientific objectives can be achieved without adhering to such constraints. Investigation of a scheduling algorithm that allows flexible time constraints shows that improvements in workstation throughput as great as 50% can be reached by modest flexibility in the timing of operations in the experiments. Several heuristics that might be used with the scheduling algorithm were tested; a heuristic that schedules long experiments while first keeping the workstation busy was shown to be a good general choice.

Algorithms↗
PubMed1994

Synthesis of amphipathic porphyrins and their photoinduced electron transfer reactions at the lipid bilayer-water interface.

A one flask synthesis of cis-substituted amphipathic porphyrins is reported. These porphyrins were used to study electrostatic effects on photoinduced electron transfer across the lipid bilayer-water interface. A neutral porphyrin undergoes only dynamic interfacial electron transfer reactions irrespective of charge of the acceptor, although ionic strength effects indicate a negative charge on the porphyrin donor species. A dianionic porphyrin forms an interfacial static complex with a dicationic electron acceptor, methyl viologen, at low ionic strength. The electron transfer rate within the complex is slow, 10(5) approximately 10(6) s-1, which is attributed to a near orthogonal orientation between the donor and the acceptor pi orbitals.

Electrochemistry↗
PubMed1992

252Cf plasma desorption mass spectrometry in the synthesis of porphyrin model systems.

252Cf plasma desorption mass spectrometry has been used in the characterization of more than 100 synthetic porphyrins ranging in mass from 614 u for tetraphenylporphyrin to over 2000 u for some porphyrin model systems. In virtually every case, 252Cf plasma desorption mass spectrometry yielded an intense ionized molecule ion [M.+ and/or (M+H)+], irrespective of the groups appended to the porphyrin. The appended groups include carboxylic acids, amides, imides, chloroacetamides, Fmoc-protected amino acids, aromatic amines, nitriles, alkynes, alkenes, esters, active esters, benzyl ethers, acetals, dithioacetals, ketones, imines, phenols, quinone, hydroquinone, ferrocene, cyanine dyes, trimethylsilyl protecting groups, nitro groups, and combinations of these functionalities. Metalloporphyrins and porphyrin-porphyrin dimers are also analyzed with ease. Resolved isotopic peaks were observed for porphyrins with molecular weights below 1000, and unresolved isotopic peaks yielding average masses were observed for porphyrin compounds with higher molecular weights. The limited resolution in the higher molecular weight range does not lessen the utility of the method because the observation of the molecule ions [M.+ and/or (M+H)+] provides unambiguous evidence concerning the success of the synthesis. The 252Cf plasma desorption mass spectra of porphyrins are not complicated by chemical transformations. This method is ideally suited for rapid analysis of synthetic porphyrins and provides a powerful tool for chemists engaged in the synthesis of complex organic molecules.

Californium↗
PubMedDate not supplied

Androgen regulation of the Pem homeodomain gene in mice and rat Sertoli and epididymal cells.

Although the role of homeodomain transcription factors during embryogenesis is well known, their developmental function in postnatal animals is only beginning to be understood. We examined the regulation and expression pattern of Pem, a homeodomain protein that may regulate androgen-dependent events in the testis and epididymis. Immunohistochemical analysis showed that Pem protein is expressed selectively in the nuclei of Sertoli cells during the androgen-dependent stage of the seminiferous epithelium cycle in vivo. RNase protection analysis revealed that a proximal promoter was responsible for androgen-dependent mouse Pem expression in testis and epididymis in vivo, whereas a distal promoter was used in placenta. The mouse Pem gene was expressed at approximately 10-fold higher levels in the testis than in the epididymis; conversely, the rat Pem gene was expressed at >10-fold higher levels in the epididymis than in the testis. Because androgen-binding protein has been proposed to transport androgens from the testis to the epididymis, we tested whether the > or = 20-fold higher levels of androgen-binding protein expression in the rat, compared to that of mouse, are responsible for the differential expression of Pem in these two rodent species. Studies with androgen-binding protein transgenic mice demonstrated that the species-specific difference in androgen-binding protein expression is unlikely to be responsible for the species-specific difference in Pem expression. We found that androgen is necessary but not sufficient for Pem expression, since purified Sertoli cells rapidly down-regulated Pem transcripts in culture, regardless of the presence of testosterone. We conclude that Pem gene expression in Sertoli cells requires other cell types or cellular factors in addition to androgen.

Androgen-Binding Protein↗