The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity.

Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 A resolution. The structure extends helix alpha4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of alpha4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop is important for a postbinding catalytic step.     

Conformational diversity of single‐stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases

Repetitive extragenic palindrome (REP)—associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT‐encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase‐bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex‐favoring 5′‐G and 3′‐C‐rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 585–596, 2015.     

A molecular analysis of the isolated rat posterior frontal and sagittal sutures: differences in gene expression

Although it is one of the most commonly occurring craniofacial congenital disabilities, craniosynostosis (the premature fusion of cranial sutures) is nearly impossible to prevent because the molecular mechanisms that regulate the process of cranial suture fusion remain largely unknown. Recent studies have implicated the dura mater in determining the fate of the overlying cranial suture; however, the molecular biology within the suture itself has not been sufficiently investigated. In the murine model of cranial suture fusion, the posterior frontal suture is programmed to begin fusing by postnatal day 12 in rats (day 25 in mice), reliably completing bony union by postnatal day 22 (day 45 in mice). In contrast, the sagittal suture remains patent throughout the life of the animal. Using this model, this study sought to examine for the first time what differences in gene expression--if any--exist between the two sutures with opposite fates. For each series of experiments, 35 to 40 posterior frontal and sagittal suture complexes were isolated from 6-day-old Sprague-Dawley rat pups. Suture-derived cell cultures were established, and ribonuicleic acid was derived from snap-frozen, isolated suture tissue. Results demonstrated that molecular differences between the posterior frontal and sagittal suture complexes were readily identified in vivo, although these distinctions were lost once the cells comprising the suture complex were cultured in vitro. Hypothetically, this change in gene expression resulted from the loss of the influence of the underlying dura mater. Significant differences in the expression of genes encoding extracellular matrix proteins existed in vivo between the posterior frontal and sagittal sutures. However, the production of the critical, regulatory cytokine transforming growth factor beta-1 was equal between the two suture complexes, lending further support to the hypothesis that dura mater regulates the fate of the overlying cranial suture.     

Effects of FSH and LH on incorporation of [3H]thymidine into follicular DNA

To assess the roles of FSH and LH on follicular growth, after various experimental manipulations, hamster follicles were sorted into 10 stages and incubated for 4 h with [3H]thymidine. Stages 1-4 correspond to follicles with 1-4 layers of granulosa cells, respectively; Stage 5 = 5 or 6 layers of granulosa cells plus theca; Stage 6 = 7-8 layers of granulosa cells plus theca; Stage 7 = early formation of the antrum; Stages 8-10 = small, intermediate and large antral follicles, respectively. Phenobarbitone sodium injected at 13:00 h on pro-oestrus blocked the normal rise of blood FSH and LH concentrations at 15:00 h and prevented the increase of [3H]thymidine incorporation into follicles of Stages 1-9. The optimal treatment to reverse the effects of phenobarbitone was 1 microgram FSH and 2 micrograms LH injected i.p. at 13:00 h which restored DNA replication to follicles of Stages 2-10: FSH acted primarily on Stages 2-5 and LH on Stages 5-10. Injection of phenobarbitone at 13:00 h on prooestrus followed by 2.5 micrograms FSH at 22:00 h restored DNA synthesis by the next morning to follicles at Stages 1-8. In hamsters hypophysectomized at 09:00 h on the day of oestrus (Day 1), injection on Day 4 of 2.5 micrograms FSH restored DNA synthesis 6 h later to Stage 2-6 follicles. Unilateral ovariectomy on Day 3 resulted 6 h later in an acute rise in FSH and LH and change of follicles from Stage 4 to Stage 5 but, paradoxically, there was decreased synthesis of DNA in follicles of Stages 5-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erratum

Costal and auricular cartilage obtained from mutant rabbits exhibiting lysozyme deficiency has been found to be identical to similar tissue from control animals in a variety of biochemical parameters. These data seriously question the putative role of lysozyme as a structural component of cartilage.     

Estrogen receptors in ovary and uterus of immature hamster and rat: effects of estrogens

Cytosolic and nuclear estrogen receptors in the ovary and uterus of immature rats and hamsters were determined to evaluate why exogenous estrogens were ineffective in stimulating follicular maturation in the hamster compared to the rat. Animals were injected sc with oil or single injection of 1 mg estradiol cyclopentylpropionate (ECP) on Day 23 or a daily injection of 2 mg diethylstilbestrol (DES) on Days 23-25 and killed on Day 26. Total binding sites for estrogen in ovarian cytosol of control hamsters were half the number in the rat ovary (28 fmole/mg protein) and about 50% of the receptors were occupied in the hamster. The apparent affinity of the estrogen-cytosol receptor complex was also lower in the hamster (Kd; 1.41 nM) than in the rat (Kd; 0.52 nM). After ECP treatment, there was a tendency for translocation in all 4 tissues examined even though some differences were not statistically significant. However, after DES treatment both cytosol and nuclear estrogen receptors decreased in both species. This discrepancy may be due to the difference in the time course of the nuclear translocation, the difference in metabolism and difference in the binding potencies of ECP and DES. The lack of ovarian responsiveness to estrogen in the hamster thus appears to be due to the reduced number of cytosol receptor sites which have a low affinity for estrogen and are already partially occupied.     

Efficient slow-growth conservation and assessment of clonal fidelity of Ullucus tuberosus Caldas microshoots

Plant Cell, Tissue and Organ Culture (PCTOC) - Slow-growth is a biotechnological tool for medium-term conservation of plant germplasm under in vitro conditions. In the present study, we assessed...     

A translocation heterozygote in garden Canna

A case of translocation heterozygosity has been discovered in gardenCanna. Morphologically the heterozygote is distinctive, being a pygmy type with small flowers which do not open. Cytologically it shows a ring or a chain of 4 plus 7 bivalents. The present observations reveal that rings disjunct usually in adjacent, and chains in an alternate manner. The subsequent course of meiosis is normal. The variety is totally sterile both after self and cross pollination. It has very likely arisen as a hybrid between two parents differing in one interchange.