Premature termination of tubulin gene transcription in Xenopus oocytes is due to promoter-dependent disruption of elongation.

We have shown previously that the Xenopus alpha-tubulin gene, X alpha T14, exhibits premature termination of transcription when injected into oocyte nuclei. The 3' ends of prematurely terminated transcripts are formed immediately downstream of a stem-loop sequence found in the first 41 bp of the 5' leader. We show here, using deleted constructs, that premature termination requires the presence only of sequences from -200 to +19 relative to the initiation site. Deletion of the stem-loop does not increase the production of extended transcripts, and premature termination apparently continues at nonspecific sites. This finding indicates that disruption of the elongation phase of transcription rather than abrogation of a specific antitermination mechanism is the cause of premature termination in X alpha T14. We also found that disruption of elongation on a reporter gene could be induced specifically by competition with X alpha T14 promoters. To identify which elements of the promoter might interact with elongation determinants to cause this competition, we constructed a series of internal promoter mutants. Most mutations in the -200 to -60 region of the promoter had some effect on initiation frequency but did not cause any significant change in levels of premature termination. However, mutations in the core promoter that removed the TATA box consensus causes major change in initiation and resulted in a marked decrease in the production of prematurely terminated transcripts relative to extended transcripts. We discuss why such promoters can apparently escape the disruption of elongation that leads to premature termination.

Premature termination of transcription can be induced on an injected alpha-tubulin gene in Xenopus oocytes.

The Xenopus laevis alpha-tubulin gene X alpha T14, which is highly expressed during oogenesis, exhibits accurate and efficient transcription initiation when microinjected into X. laevis oocytes. However, we found previously in nuclease protection assays of transcripts from injected X alpha T14 that many protected fragments that were shorter than expected could be produced. We show here by exonuclease VII mapping, Northern (RNA) blotting, and gel fractionation of RNA that these fragments were caused by truncated transcripts that share the same initiation sites as mature transcripts but whose 3' ends are located in the 5' leader just 45 to 72 nucleotides downstream. We present evidence from the analysis of in vitro pulse-labeled RNA that these truncated transcripts are formed by premature transcription termination rather than by RNA processing. At low template levels, very little premature termination occurred, but as more DNA was injected, the proportion of transcripts that were prematurely terminated increased steadily, even at template levels at which the initiation machinery was unsaturated. At high template levels, most transcripts were prematurely terminated. These results suggest that some sort of saturable antitermination function operates in oocytes in a manner that is dependent on the number of appropriate templates available rather than on the number of polymerases that initiate transcription. They also suggest that measures of initiation frequency may not always be a reliable means of assessing the amount of transcription of injected genes in oocytes.

Identification in the human genome of mobile elements spread by DNA-mediated transposition.

We have identified in the human genome two families of mobile elements possessing the sequence characteristics of transposons that move directly from DNA to DNA rather than requiring the reverse transcription of an RNA intermediate. One type of element is closely related to the autonomous transposable element, mariner, and comprises a coding region for a transposase protein flanked by short terminal inverted repeat sequences (TIRs) of 31 or 32 bp. Elements of the second type form a family of short interspersed repetitive elements (SINEs) that are composed simply of two 37 bp TIRs surrounding six unique bps. The TIRs of the human mariner family are identical in all but one position to those of the SINE family, suggesting that the inverted-repeat SINEs represent non-autonomous transposable elements dependent on mariner-type transposase for mobility. Evidence for the mobility of both types of element is provided by examples of their integration into other repeat sequences and by the comparison of orthologous sites in cattle and human genomes. This evidence also shows that these elements have been active in DNA-mediated transposition at some point in the mammalian lineage. Therefore, it appears that the process of DNA-mediated transposition has occurred in mammalian cells and that its maximal cis-requirements are contained in the 80 bp consensus sequence of the human inverted-repeat SINE family.

A novel group of families of short interspersed repetitive elements (SINEs) in Xenopus: evidence of a specific target site for DNA-mediated transposition of inverted-repeat SINEs.

We have isolated from Xenopus borealis members of a family of short interspersed repetitive elements (SINEs) that we have termed Xbr. Xbr elements are also present in other Xenopus genomes and are typically framed by 46 bp terminal inverted repeats (TIRs). These TIRs and those of two previously described families of inverted-repeat SINEs from X. laevis begin with the sequence TTAAAGGRR. Knowledge of this consensus, termed the T2 motif, allowed us to define four previously uncharacterized families of inverted-repeat SINEs from Xenopus database sequences. We estimate that the group of seven SINE families that possess the T2 motif accounts for about 10% of all X. laevis SINEs. Novel evidence for the transposition of inverted-repeat SINEs is provided: (1) by examples of the presence/absence of T2 elements at corresponding locations in either duplicated genes or pseudotetraploid gene homeologues; and (2) by the existence of contiguous elements from different T2 families that are joined precisely by their TIRs. These examples provide novel evidence for a DNA-mediated mechanism of T2 element transposition. They also show that the tetranucleotide, TTAA, which flanks integrated elements on both sides and is present once at unoccupied sites, is the obligate target site for T2 insertion. The use of a specific sequence as a target site for SINE insertion is unexpected, although such specificity is exhibited by a limited number of larger transposable elements that encode their own transposase. The clear evidence for DNA-mediated transposition provided by T2 elements demonstrates that the evolution and maintenance of SINE families in vertebrate genomes results from two distinctive mechanisms.

Conservation of intergenic spacer length in ribosomal DNA of the tailed frog, Ascaphus truei.

We have examined the organization of cloned rDNA [encoding ribosomal RNA (rRNA)] repeat units from the tailed frog, Ascaphus truei, and have compared rDNA spacer lengths in the genomes of eleven individuals from two widely-separated populations. This comparison has shown that the A. truei spacer is always very short (about 1.5 kb) and that it is remarkably constant in length. In none of the individuals tested were more than two spacer-length classes found and the maximum difference in spacer length found in comparisons both within single animals and across both populations was about 120 bp. We point out those structural features that may contribute to the unusual stability of this spacer and the consequent absence of the extensive length heterogeneities found amongst rDNA repeat units in most genomes.

Alterations in cloned Xenopus ribosomal spacers generated by high-frequency plasmid recombination.

In an attempt to understand the mechanisms that have led to the complex intraspecific heterogeneity in spacer organisation found in Xenopus r.DNA, we have transformed Escherichia coli strains that exhibit high levels of plasmid recombination with Xenopus laevis r.DNA sequences inserted in pBR322. We have found that in these recBC sbcA strains, plasmid recombination results in the generation of many types of altered r.DNA spacer at a high frequency. The altered spacers result from two types of deletion/duplication events that occur in the promoter-derived sequences making up much of the spacer. The alterations caused by these events mimic in several ways the different types of organisation found among spacers in the genome. The sbcA-dependent plasmid recombination apparently provides a test system for reconstructing some of the recombinational events that operate during the evolution and maintenance of spacer DNA sequences in vivo.

Stimulation of RNA 3' processing by flanking DNA in Xenopus oocytes.

We have previously shown that the second poly(A) signal of the Xenopus laevis alpha-tubulin gene X alpha T14, which contains the rare hexanucleotide CAUAAA, requires a surprisingly large amount of 3' flanking DNA to be used efficiently in Xenopus oocytes. To investigate the nature of the interaction between the X alpha T14 3' flank and upstream 3' processing sites, we have developed a modified oocyte assay based on the stimulation of processing at a single poly(A) signal. We mutated both the hexanucleotide and GU/U-rich components of a strong synthetic poly(A) signal (SPA) in order to weaken it severely. We found that efficient use of the mutant signal could be fully restored by the addition of 1.2 kb of X alpha T14 3' flank, but only in its natural orientation. Functional dissection of the X alpha T14 3' flank defined two separate regions that were each capable of partially restoring processing efficiency, presumably because they contain multiple, relatively weak processing enhancers. We discuss how the stimulation of 3' processing by flanking regions in oocytes could be explained by mechanisms that operate on the processing machinery directly or by indirect effects mediated by transcriptional pausing.

Clinical aspects of vasectomies performed in the United States in 1995.

OBJECTIVES: Currently, no surveillance system collects data on the numbers and characteristics of vasectomies performed annually in the United States. This study provides nationwide data on the numbers of vasectomies and the use of no-scalpel vasectomy, various occlusion methods, fascial interposition, and protocols for analyzing semen after vasectomy. METHODS: A retrospective mail survey (with telephone follow-up) was conducted of 1800 urology, family practice, and general surgery practices drawn from the American Medical Association's Physician Master File and stratified by specialty and census region. Mail survey and telephone follow-up yielded an 88% response rate. RESULTS: In 1995, approximately 494,000 vasectomies are estimated to have been performed by 15,800 physicians in the United States. Urologists performed 76% of all vasectomies, and nearly all (93%) urology practices performed vasectomies in 1995. Nearly one third (29%) of vasectomies in 1995 were no-scalpel vasectomies, and 37% of physicians performing no-scalpel vasectomies taught themselves the procedure. The most common occlusion method in 1995 (used for 38% of all vasectomies) was concurrent use of ligation and cautery. In 1995, slightly less than half (48%) of all physicians surveyed interposed the fascial sheath over one end of the vas when performing a vasectomy. Protocols for ensuring azoospermia varied: 56% of physicians required one postvasectomy semen specimen; 39% required two, and 5%, three or more. CONCLUSIONS: No-scalpel vasectomy, used by nearly one third of U.S. physicians, has become an accepted part of urologic care. Physicians' variations in occlusion methods, use of fascial interposition, and postvasectomy protocols underscore the need for large scale, controlled, and statistically valid studies to determine the efficacy of occlusion methods and fascial interposition, as well as whether azoospermia is the only determination of a successful vasectomy.

The time course of male meiosis in the red-backed salamander, Plethodon cinereus.

Tritiated-thymidine autoradiography was used to follow the progress of cells through meiosis in male P. cinereus at 20 degrees C. Spermatocytes spend 7 days in leptotene, 5 days in zygotene and 3 days in pachytene before entering the diffuse stage. Diffuse lasts for 8 days and is followed by a diplotene of 2 days. First and second meiotic metaphase occur a total of 26 days after the end of premeiotic S. Considering the information for P. cinereus together with that for 3 other species, it appears that in amphibians the duration of meiosis does not show the linear relation with C-value that has been described for other groups of organisms.

Short interspersed repeats from Xenopus that contain multiple octamer motifs are related to known transposable elements.

We have identified in an intron of an X. laevis alpha-tubulin gene a member of a novel family of short (226-431 bp) interspersed repetitive elements. We have isolated other members of this family, which we term Ocr, from ovary cDNA and genome libraries and have identified another two in the published sequences of an H1B histone gene cluster and an actin gene intron. The termini of the Ocr elements are formed by a 19 bp inverted repeat that has clear sequence homologies to those of certain large transposable elements, such as 1723 (Xenopus) and Ac (maize). However, the Ocr elements do not appear to be deletion derivatives of larger transposons. The internal regions of the Ocr elements contain multiple copies of the octamer motif (ATTTGCAT) arranged as divergently-orientated dyads. We have shown by a gel mobility shift assay that these octamer dyads specifically bind what is presumably an OTF-type activator protein in oocyte nuclear extracts. We speculate that short interspersed repetitive families of this type may be generated by a mechanism of replicative transposition that uses a DNA intermediate and involves the interaction of DNA-binding proteins also utilised in other cellular processes.

Alternative 3' processing of Xenopus alpha-tubulin mRNAs; efficient use of a CAUAAA polyadenylation signal.

The Xenopus laevis alpha-tubulin gene X alpha T14 produces two mRNAs of 1.7 and 2.15 kb and we have shown that this is due to the use, at approximately equal frequency, of alternative 3' processing sites. Unusually, the hexanucleotide polyadenylation signal responsible for use of the downstream site, pA2, is CAUAAA in contrast to the consensus AAUAAA used at the upstream site, pA1. Since such a variant hexanucleotide would normally be expected to reduce drastically the efficiency of 3' processing, we have examined the 3' flanking sequences involved in pA2 usage in injected oocytes. In deletion mutants with 40 bp or 440 bp of 3' flanking DNA use of pA2 was almost totally abolished whereas when 770 bp of the natural flank was present pA2 was used normally. This polyadenylation signal therefore requires an unexpectedly large amount of flanking DNA and we have identified in the required region a member of a novel family of 450 bp interspersed repeats that we have termed Pir elements. We speculate that because of the variant hexanucleotide efficient use of pA2 has to be potentiated by the Pir element, perhaps through an effect on transcriptional pausing or termination.

An oocyte-expressed alpha-tubulin gene in Xenopus laevis; sequences required for the initiation of transcription.

We have studied the expression of X alpha T14, a member of the alpha-tubulin multigene family in Xenopus laevis. Small amounts of X alpha T14 RNA are detectable in a range of cell types, but much higher levels are present in ovary and tissue culture cells. In oocytes X alpha T14 transcripts accumulate during early vitellogenesis but their level declines in more advanced stages. Faithful and efficient initiation of transcription occurred on cloned X alpha T14 injected into oocytes even at low template levels. We have examined the amount of transcript produced by various deletion mutants relative to a co-injected control gene. The presence of 200bp of DNA 5' and 53bp of DNA 3' to the initiation site sufficed for high levels of promoter activity, although maximum activity required 560 bp of 5' flanking DNA. The DNA between -200 and -60 was necessary for transcription in oocytes and contains several sequence motifs implicated in transcriptional regulation including three CCAAT boxes and a sequence resembling a heat shock element. An 8 bp deletion that removed the latter element from 5kb of 5'-flanking DNA reduced promoter activity by 60%.

Identification of novel genes encoding transcription elongation factor TFIIS (TCEA) in vertebrates: conservation of three distinct TFIIS isoforms in frog, mouse, and human.

We report the characterization of cDNA clones that define a new, third isoform of the transcription elongation factor TFIIS in Xenopus, mouse, and human. In Xenopus the mRNA of this isoform, termed TFIIS.h, shows tissue-restricted expression, frequently contains unspliced introns, and is characterized by three near-perfect 150-bp repeats at the 5'-terminus. Although we were unable to isolate full-length cDNAs, it is clear that these repeats contain an open reading frame encoding a region of TFIIS.h that is much more complex than in other isoforms. Identification of ESTs encoding TFIIS.h in mouse and human followed by the sequencing of cognate cDNA clones enabled the complete TFIIS.h coding region to be predicted. The conserved N- and C-terminal domains of mammalian TFIIS.h (TCEA3) are separated by a linker region that is more variable in sequence and that is also 50 amino acids longer than in other isoforms. The repetitive region of Xenopus TFIIS.h apparently corresponds to an even more extended linker. Phylogenetic analysis of TFIIS sequences demonstrates the ancient origins of the three vertebrate isoforms, although they appeared functionally equivalent in in vitro RNA cleavage assays.

Ovary-specific expression of a gene encoding a divergent alpha-tubulin isotype in Xenopus.

We are investigating the structure and regulation of alpha-tubulin genes expressed in amphibian oocytes. We have characterised here a gene, X alpha T207, that produces a major alpha-tubulin mRNA of Xenopus laevis ovary. X alpha T207 mRNA was not detected in other frog tissues and its production may therefore be a key identifying feature of ovarian differentiation. In comparison to the tubulin isotypes so far described in mammals and Xenopus, the alpha-tubulin encoded by X alpha T207 is divergent in overall amino acid sequence, particularly in the N-terminal region between residues 39-50. This pattern of divergence is also displayed by the ovary-specific alpha-tubulin gene of Drosophila, D alpha 4, although the two genes do not appear to be orthologous. The development of specialised microtubular structures and activities in oocytes, eggs and early embryos may then be correlated with the expression of a divergent alpha-tubulin isotype in a wide range of organisms. To understand the basis of the ovary-specific expression of X alpha T207 we examined the transcriptional activity of wild type and mutant promoters after their microinjection in Xenopus oocytes. Only 65 bp upstream of the initiation site were required for full activity of the X alpha T207 promoter, and an element fitting the Y-box consensus was involved in controlling the efficiency of initiation. Previous oocyte injection experiments have implicated the Y-box in the oocyte-specific transcription of genes that are also expressed in other cell types, so its involvement in the oocyte-restricted expression of X alpha T207 further suggests that transcription factors recognising the Y-box normally regulate gene expression during oocyte development. Since a Y-box also occurs in the D alpha 4 promoter, our results suggest that in both organisms oocyte-specific expression of a divergent alpha-tubulin could be achieved by a common mechanism.

Multiple ribosomal gene sites revealed by in situ hybridization of Xenopus rDNA to Triturus lampbrush chromosomes.

A variety of 3H-labelled ribosomal gene probes were hybridized in situ to the nascent transcripts of lampbrush chromosomes from the crested newt, Triturus cristatus carnifex. The probes were from Xenopus laevis and included rDNA isolated by CsCl gradient centrifugation, recombinant plasmids and purified restriction fragments of rDNA. All the probes gave essentially the same result. About 10-15 loop pairs were distinctly labelled in each preparation, almost all of them located on the heteromorphic arms (HTAs) of chromosome 1. Ribosomal gene probes were also hybridized in situ to the DNA of denatured mitotic chromosomes from some of the individuals used to provide lampbrush preparations. Minor, scattered sites of hybridization were found in the HTAs, but the main clusters of ribosomal genes were found on chromosomes 6 and/or 9, in agreement with previous determinations of nucleolus organizer position in this species. However, the nucleolus organizers were not sites of labelled loops in lampbrush transcript hybridizations.--We have incubated isolated lampbrush-stage nuclei in media containing alpha-amanitin and labelled RNA precursors. Although extrachromosomal nucleolar genes incorporated label, supposedly due to transcription by RNA polymerase I, no lampbrush loops were labelled.--It appears that in T. c. carnifex there are ribosomal gene sequences at the main nucleolus organizers and at a number of sites scattered along the HTAs. The ribosomal genes at the nucleolus organizers are not extended in the form of actively transcribing loops unlike the ribosomal sequences on the HTAs, which are heavily labelled in transcript hybridization. The ribosomal sequences on the HTAs appear not to be transcribed by the same RNA polymerase that transcribes the ribosomal genes of extrachromosomal nucleoli.

Genes encoding isoforms of transcription elongation factor TFIIS in Xenopus and the use of multiple unusual RNA processing signals.

We have identified cDNAs encoding three related forms of transcription elongation factor TFIIS (S-II) in Xenopus laevis ovary. Comparison of Xenopus and mammalian sequences identifies likely diagnostic amino acids that distinguish classes of vertebrate TFIIS. The diversity of TFIIS polypeptides in Xenopus is due partly to the presence of two diverged genes in this tetraploid genome. We isolated genomic clones containing one of the genes, xTFIIS.oA, and, unlike a previously described vertebrate TFIIS gene, found that it contains introns. Alternative splicing at a CAG/CAG motif containing the 3' splice site of intron 4 produces the third form of xTFIIS, which differs from one of the others simply in lacking Ser109. Intron 6 of xTFIIS.oA contains splice and branch site consensus sequences conforming to those of the minor class of AT-AC introns and this was confirmed for the homeologous xTFIIS.oB gene by genomic PCR. Other unusual but functional variants of RNA processing signals were found in xTFIIS genes at the 5' splice site of intron 8 and the polyadenylation hexanucleotides. Utilization of multiple unusual processing signals may make the generation of mature xTFIIS.o mRNAs inefficient and the possible regulatory consequences of this are discussed.

Transcription in cloned spacers of Xenopus laevis ribosomal DNA.

Rare individuals of Xenopus laevis exhibit frequent initiation of transcription in the spacers of oocyte ribosomal DNA (rDNA). Using electron microscopy we have characterized spacer transcription in such an individual and have confirmed that the sites of transcription initiation correspond to the imperfectly duplicated promoters ("Bam islands") present in the X. laevis rDNA spacer. We have cloned a repeat unit containing a gene and a spacer from this individual and have injected the recombinant plasmid, pXlr 164, into oocytes of other X. laevis individuals. In electron microscope preparations the spacers of some of the cloned repeats were transcribed by RNA polymerase I. This demonstrates that the ability to initiate transcription at the Bam islands is a property of the spacer DNA. On pXlr 164, initiation in the spacer occurred about 5% as frequently as transcription from the gene promoter. However, transcribed spacers were as closely packed with RNA polymerase as was the gene. We conclude that polymerase I promoters may vary over a wide range in the frequency with which they "activate" but that once activated all can load polymerases to maximal density. The presence or absence of spacer transcription had no observable effect on either the frequency of activation or the density of polymerase loading of the gene immediately downstream. A subclone, pXlr 264, containing only spacer DNA also showed regular initiation and termination, providing further evidence that there is an effective "fail-safe" termination signal 225 base pairs upstream from the rRNA gene initiation site.

Condom use and partner characteristics among young adult males in urban Ghana, aged 15-24.

Addressing male sexual behavior and condom use is a high priority for adolescent health programs. Using the 1997 Ghana Psychographic Survey, the aim of this study is to explore the factors related to current, partner-specific condom use, by Ghanaian males aged 15-24 years. A multivariate regression analysis reveals an independent association between psychographic attitudes, peer network characteristics, sexual partner characteristics, and some level of condom use with a nominated sexual partner. The most important predictor for both condom use consistently as well as condom use at least sometimes was respondents' knowing someone who died as a result of AIDS. This finding suggests that future interventions should aim to personalize the risk of HIV/STIs, promote condom use with a range of partner types, and educate youth about the importance of consistent use.

Variations in transcriptional activity of rDNA spacer promoters.

We have compared the DNA sequences of several different examples of the duplicated polymerase I promoters that are found in rDNA spacers of Xenopus laevis. Although different spacers exhibit different amounts of transcription in vivo, this does not seem to be due to DNA sequence differences between spacer promoters. We have found that several different spacer promoters when subcloned and injected into oocytes exhibit similar promoter activities when transcription is assayed by primer extension analysis. Moreover, the activity of these spacer promoters is the same as that of a co-injected gene promoter. The equivalence of spacer promoter activity and gene promoter activity was also found when rDNA plasmids containing intact spacers were injected into oocytes and transcription assayed by primer extension. This is in contrast to (1) the inactivity normally exhibited by the promoters of endogenous spacers in oocytes, (2) the relative inactivity of spacer promoters found when transcription of the same rDNA plasmids is assayed by electron microscopy.

RNA polymerase II in Cajal bodies of amphibian oocytes.

Cajal bodies (coiled bodies) are nuclear organelles that contain a variety of components required for transcription and processing of RNA. Cajal bodies in amphibian oocytes are stained by mAb H14, which recognizes the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II when the heptapeptide repeat is phosphorylated on serine-5. Oocytes were treated with the transcription inhibitor 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), which prevents phosphorylation of the CTD. Cajal bodies from oocytes that had been treated for 2-3 h with DRB no longer stained with mAb H14, but staining reappeared when the inhibitor was washed out. Epitope-tagged transcripts of two small subunits of polymerase II, RPB6 and RPB9, were injected into the cytoplasm of Xenopus and Triturus oocytes. Newly translated RPB6 and RPB9 were specifically targeted to Cajal bodies within 4 h, and Cajal bodies remained the site of highest concentration of tagged protein during the next 2 days. These data suggest that polymerase subunits pass through the Cajal bodies with a transit time no greater than a few hours. We discuss the possibility that Cajal bodies are sites of assembly or modification of the transcription machinery of the nucleus.