David A. Wright

Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III.

Whereas the genes coding for trimethyl guanosine‐capped snRNAs are transcribed by RNA polymerase II, the U6 RNA genes are transcribed by RNA polymerase III. In this study, we have analyzed the cis‐regulatory elements involved in the transcription of a mouse U6 snRNA gene in vitro and in frog oocytes. Transcriptional analysis of mutant U6 gene constructs showed that, unlike most known cases of polymerase III transcription, intragenic sequences except the initiation nucleotide are dispensable for efficient and accurate transcription of U6 gene in vitro. Transcription of 5′ deletion mutants in vitro and in frog oocytes showed that the upstream region, within 79 bp from the initiation nucleotide, contains elements necessary for U6 gene transcription. Transcription studies were carried out in frog oocytes with U6 genes containing 5′ distal sequence; these studies revealed that the distal element acts as an orientation‐dependent enhancer when present upstream to the gene, while it is orientation‐independent but distance‐dependent enhancer when placed down‐stream to the U6 gene. Analysis of 3′ deletion mutants showed that the transcription termination of U6 RNA is dependent on a T cluster present on the 3′ end of the gene, thus providing further support to other lines of evidence that U6 genes are transcribed by RNA polymerase III. These observations suggest the involvement of a composite of components of RNA polymerase II and III transcription machineries in the transcription of U6 genes by RNA polymerase III.

Genetics and Ontogeny of -Glycerophosphate Dehydrogenase Isozymes in Drosophila melanogaster

Abstractα-Glycerophosphate dehydrogenase (GPDH) occurs in Drosophila melanogaster in three isozymic forms. These are separable by starch gel electrophoresis and have been tentatively numbered 1, 2, and 3. GPDH-1 is most concentrated in the adult thorax and GPDH-3 in the abdomen; 1 and 3 are in approximately equal amounts in the head. GPDH-2 is relatively weak in all preparations. In larvae, only GPDH-3 is present. Purified GPDH-1 has optimal activity at pH 6.7–7.0. GPDH-3 at pH 7.5, and GPDH-2 is intermediate. Changes in total GPDH activity parallel larval growth, pupal histolysis, and differentiation of adult tissues. In the latter period the ratio of activity at pH 6.7 to pH 7.6 increases, reflecting the shift from GPDH-3 to GPDH-1. Two types of homozygous GPDH patterns which differ in the electrophoretic mobilities of all three isozymes have been found in inbred strains. In heterozygous adults six bands, the parental forms of GPDH-1 and GPDH-3 and “hybrid” forms of each, can be resolved. Analysis of F2 and backcross progeny suggests that a single genetic locus affects all three isozymes. Heterozygous embryos have only the maternal form of GPDH-3 until just before they hatch as first instar larvae. At this stage they have maternal and paternal GPDH-3 plus an intermediate band.

Nuclear and cytoplasmic contributions to dehydrogenase phenotypes in hybrid frog embryos

Abstract Frogs in the Rana pipiens group including R. palustris and geographic races of R. pipiens form viable diploid hybrids which develop through metamorphosis. There are differences in electrophoretic mobilities of several enzymes among the frogs used for hybridization. After electrophoresis of extracts of hybrid frog embryos on starch gel the maternal, paternal, and possible hybrid molecules are detected with appropriate specific enzyme staining. In early embryos only the maternal type enzymes are present. In viable diploid hybrids the paternal form and a ‛hybrid” molecule of 6-phosphogluconate dehydrogenase (6PGD) are detected by stage 17 ½ (late tail bud). Similarly, evidence of gene expression for the B subunit of lactate dehydrogenase (LDH) is seen at stage 18 ½ (muscular movement). Expression of the gene for soluble isocitrate dehydrogenase (IDH-s) is seen by stage 19 (heart beat). A nuclear gene controls mitochondrial malate dehydrogenase (MDH-m), and its expression is also evident by stage 19 heart beat). A maternal effect on each of the enzyme phenotypes persists even after these events; in the case of LDH and MDH, until after stage 25 (feeding tadpole). Androgenetic haploid hybrids show a range of development. The homologous combinations show the typical haploid syndrome but develop to the feeding stage. Heterologous combinations show more severe symptoms; some arrest at neurula stages, but others develop through hatching. In androgenetic haploid hybrids the paternal (nuclear) forms of the dehydrogenases studied appear at the same time as the diploid controls. This occurs even though the haploids are grossly abnormal and retarded in morphogenesis. Since the maternal genome was not present in such haploid embryos, the maternal effect on enzyme phenotypes clearly must be cytoplasmic in origin. No hybrid enzymes (composed of both maternal and paternal type subunits) are detected in androgenetic haploid hybrids. This and other evidence is consistent with the idea that the maternal effects are due to enzymes present in the egg prior to fertilization, not to continued synthesis directed by stable messenger RNA. It also indicates that the degradation of maternal (cytoplasmic) enzymes in vivo does not yield subunits capable of reaggregation with newly synthesized subunits to form active enzymes.

Time of Expression of Genes Controlling Specific Enzymes in Drosophila Embryos

The activities of several enzymes were determined in homogenates of Drosophila melanogaster embryos and early larvae. The dehydrogenases of isocitrate (IDH), α-glycerophosphate (αGPDH), glucose 6-phosphate (G6PD), and 6-phosphogluconate (6PGD) show no increase in activity until late in the embryonic period (about 20 hr). The increase in the activity of these enzymes begins before hatching and continues into the larval period. Embryos heterozygous at loci controlling specific enzymes, IDH, αGPDH, G6PD, and ADH (alcohol dehydrogenase), have only the maternal form of each enzyme throughout most of the embryonic period. Paternal and hybrid forms of the enzymes are found at hatching. This corresponds to the increase in total activity for each of the enzymes. The results are discussed in terms of the control of gene expression in development.

Partial purification and characterization of mitotic factors from HeLa cells

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.

Chromosome-bound mitotic factors: release by endonucleases

Additional evidence is presented to support our recently reported conclusion that the mitotic factors of mammalian cells, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus laevis oocytes, are localized on metaphase chromosomes. Chromosomes isolated from mitotic HeLa cells were further purified on sucrose gradients and digested for varying periods with either the micrococcal nuclease or DNase II. At each time point of digestion the amount of mitotic factors released was determined by injecting a supernatant of these fractions, obtained by high-speed centrifugation, into oocytes. The amount of DNA rendered acid soluble under the conditions of digestion used was 3% ot 5% of the total chromosomal DNA. The extent of release of mitotic factors with both nucleases was estimated to be about 30% to 40% as evidenced by the reextraction of the undigested chromosomal pellet with 0.2 M NaC1. Similar results were obtained when nuclei from G2 cells were digested under identical conditions. The release of these chromosome-bound mitotic factors by mild digestion with these nucleases though only partial, clearly demonstrates that a significant proportion of these factors are localized on metaphase chromosomes.

Efficacy and Safety of 48 Weeks of Enfuvirtide 180 mg Once-Daily Dosing Versus 90 mg Twice-Daily Dosing in HIV-Infected Patients

Abstract Purpose: To evaluate the safety and antiviral activity of once-daily (qd) enfuvirtide (ENF) compared with twice daily (bid) ENF. Method: T20-401 was a phase 2, open-label, randomized, 48-week pilot study comparing ENF 180 mg qd versus ENF 90 mg bid, added to an optimized background (OB) regimen. Patients were randomized 1:1 to receive ENF 180 mg qd given as two 90-mg injections (n = 30) or one 90-mg injection bid (n = 31), plus OB. The primary efficacy endpoint was the proportion of patients achieving a HIV-1 RNA viral load <400 copies/mL. Adherence, pharmacokinetics, safety, and tolerability parameters, including injection site reactions (ISRs), were compared between treatment arms. Results: At Week 48, 23.3% of patients on once daily versus 22.6% on twice daily (p = .969) reached <400 copies/mL and 13.3% and 22.6% (p = .323), respectively, reached <50 copies/mL. The proportion reporting ≥1 adverse event or ISRs was comparable between arms, despite an increased incidence of grade 4 erythema (13% vs. 0%), induration (33% vs. 12%), and ecchymosis (7% vs. 0%) on twice daily versus once daily. ENF adherence (≥95% of prescribed doses) was higher on once daily (80.0%) versus twice daily (58.1%). Conclusion: The antiviral efficacy, safety, and tolerability of 180 mg ENF qd appeared comparable with that of 90 mg ENF bid at Week 48, although the study was not powered to demonstrate the noninferiority of once daily versus twice daily.

Immunochemistry of frog lactate dehydrogenase (LDH) and the subunit homologies of amphibian LDH isozymes

Abstract 1. 1. The immunochemical properties of Rana pipiens lactate dehydrogenase (LDH) isozymes support the assignment of subunit structure of LDH-1 as B 4 , LDH-3 as A 2 B 2 , LDH-4 as A 3 B and LDH-5 as A 4 . 2. 2. The subunit designations are homologous with those used for avian isozymes as shown by immunochemical cross-reaction. 3. 3. The apparent reversal of tissue distribution of Taricha LDH isozymes is due to a reversal of net charge on the Taricha subunits homologous to the Rana A and B subunits.

Effects of haploidy and hybridization on the activities of four dehydrogenases in frog embryos.

Abstract Total activities of four enzymes, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase, and 6-phosphogluconate dehydrogenase were measured in diploid and androgenetic haploid embryos resulting from crosses of Rana pipiens pipiens and Rana pipiens sphenocephala . Developmental curves of these enzyme activities were compared with the DNA content of the embryos. The results suggest that decreased total enzyme activity in abnormal androgenetic haploid hybrids is due to the effects of reduced cellular proliferation and cellular degradation caused by a general nucleocytoplasmic incompatibility, not to the factors directly affecting the synthesis of each enzyme.

Role of Nonhistone Protein Phosphorylation in the Regulation of Mitosis in Mammalian Cells

The postsynthetic modification of proteins via reversible phosphorylation-dephosphorylation by phosphoprotein kinases and phosphatases has been reported to be an important mechanism in the regulation of numerous intracellular events (15,26,30). A strong correlation between the phosphorylation of histone H1 and (H3) and chromosome condensation and initiation of mitosis has been shown by several investigators (7,8,11,13,14,18,21,23). More recently, it was shown that histone H1 is also phosphorylated during premature chromosome condensation (8,22,27). However, many investigators believe that the superphosphorylation of histone H1 alone is not sufficient for chromosome condensation (10,20,22,27,38). Furthermore, chromosome decondensation could still occur even when dephosphorylation of histone H1 was blocked in mitotic cells (38). There is increasing evidence to suggest that phosphorylation of nonhistone proteins (NHP) may also be required for mitosis-related events. For example, phosphorylation of high mobility group (HMG) proteins has been suggested to be responsible for the shutting off of gene transcription during mitosis (12,28,34), increased phosphorylation of intermediate filament proteins like vimentin at mitosis (17,33), phosphorylation and dephosphorylation of laminar proteins have been implicated in the dissolution and reformation of the nuclear envelope (19,25).