Gloria Y. Lee

Nucleotide sequences that signal the initiation of transcription and translation inBacillus subtilis

SummaryWe have determined the nucleotide sequence of twoBacillus subtilis promoters (veg andtms) that are utilized by the principal form ofB. subtilis RNA polymerase found in vegetative cells (σ55-RNA polymerase) and have compared our sequences to those of several previously reportedBacillus promoters. Hexanucleotide sequences centered approximately 35 (the “-35” region) and 10 (the “-10” region) base pairs upstream from theveg andtms transcription startpoints (and separated by 17 base pairs) corresponded closely to the consensus hexanucleotides (TTGACA and TATAAT) attributed toEscherichia coli promoters. Conformity to the preferred -35 and -10 sequences may not be sufficient to promote efficient utilization byB. subtilis RNA polymerase, however, since three promoters (veg, tms andE. coli tac) that conform to these sequences and that are utilized efficiently byE. coli RNA polymerase were used with highly varied efficiencies byB. subtilis RNA polymerase.We have also analyzed mRNA sequences in DNA located downstream from eightB. subtilis chromosomal and phage promoters for nucleotide sequences that might signal the initiation of translation. In accordance with the rules of McLaughlin, Murray and Rabinowitz (1981), we observe mRNA nucleotide sequences with extensive complementarity to the 3′ terminal region ofB. subtilis 16S rRNA, followed by an initiation codon and an open reading frame.

The microtubule binding domain of tau protein

Tau protein is a microtubule-associated protein implicated in the spatial and temporal specification of microtubules and has been found in the neurofibrillary tangles of Alzheimers disease. Determination of tau protein structure has revealed three 18 amino acid repeated sequences hypothesized to be tubulin binding sites. Using tau cDNA clones from human fetal brain, we employed E. coli expression systems to synthesize tau protein and fragments of tau protein in order to identify the microtubule binding site. A fragment containing the three repeated sequences binds microtubules, while the amino-terminal half of the protein does not bind. Fragments containing two or one repeat are also capable of binding, indicating that the basic tubulin interacting unit is one repeat.

AMPA/Kainate Antagonist LY293558 Reduces Capsaicin-evoked Hyperalgesia but Not Pain in Normal Skin in Humans

Background Animal studies suggest that [small alpha, Greek]‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid‐kainate (AMPA‐KA) receptors are involved in pain processing. The effects of the competitive AMPA‐KA antagonist LY293558 in two types of experimental pain in human volunteers, brief pain sensations in normal skin, and mechanical allodynia‐pinprick hyperalgesia were studied after the injection of intradermal capsaicin. Methods Brief intravenous infusions of the competitive AMPA‐KA antagonist LY293558 were given to 25 healthy volunteers to examine acute toxicity and analgesic effects. Fifteen volunteers then entered a double‐blinded, three‐period crossover study. In a Phase II study, LY293558 infusions (100% maximally tolerated dose vs. 33% maximally tolerated dose vs. placebo) began 10 min after intradermal injection of 250 [micro sign]g capsaicin in volar forearm. Spontaneous pain, areas of mechanical allodynia and pinprick hyperalgesia, and side effects were determined every 5 min for 60 min. Results The median maximally tolerated dose was 1.3 +/− 0.4 (range, 0.9‐2.0) mg/kg. Tests of cognitive and neurological function were unchanged. Dose‐limiting side effects were hazy vision in 95% of volunteers and sedation in 40%. There were no significant changes in electrical or warm‐cool detection and pain thresholds or heat pain thresholds. LY293558 had little effect on brief pain sensations in normal skin. Both high and low doses of LY293558 significantly reduced pain intensity, pain unpleasantness, and the area in which light brush evoked pain after intradermal capsaicin. There was a trend toward a dose‐response effect of LY293558 on the area in which pinprick evoked pain after intradermal capsaicin, which did not reach statistical significance. Conclusions The authors infer that AMPA‐KA receptor blockade reduces the spinal neuron sensitization that mediates capsaicin‐evoked pain and allodynia. The low incidence of side effects at effective doses of LY293558 suggests that this class of drugs may prove to be useful in clinical pain states.

Non-motor microtubule-associated proteins

This past year, the structure and function of microtubule-associated proteins (MAPs) have been investigated in studies probing their phosphorylation, patterns of expression, and the function of the microtubule-binding domain. Cellular studies have also contributed new insights into the roles of these proteins in process outgrowth.

Effects of the 2-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid/kainate antagonist LY293558 on spontaneous and evoked postoperative pain

Previous studies suggest that 2‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐proprionic acid (AMPA)/kainate antagonists reduce experimentally induced pain. There have been no studies of AMPA/kainate antagonists in clinical pain.

Nucleotide sequence of a promoter recognized by Bacillus subtilis RNA polymerase.

SummaryWe report the nucleotide sequence of a promoter recognized by RNA polymerase from the gram-positive bacterium Bacillus subtilis. This promoter, which was isolated from B. subtilis phage SP01 DNA, is homologous to promoters for Escherichia coli RNA polymerase; the sequences of the “-35 region” and the “Pribnow box” were 5′TTGACT and 5′CATAAT, respectively (T is the thymine analog 5-hydroxymethyluracil in SP01 DNA). These sequences each differed by only a single base pair from the preferred sequences for E. coli promoters. Not surprisingly, the SP01 promoter was actively transcribed in vitro by E. coli RNA polymerase as well as by B. subtilis RNA polymerase.

Conserved nucleotide sequences in temporally controlled bacteriophage promoters

Abstract Gene expression at a middle time in the lytic cycle of Bacillus subtilis bacteriophage SP01 is controlled by the product of phage gene 28 (gp28). gp28 is a sigma-like regulatory protein that directs the bacterial core RNA polymerase to bind and initiate transcription from promoter sites for phage middle genes. Here we report on the location, orientation and nucleotide sequences of five promoters in a cluster of middle genes on the phage genome. (The nucleotide sequences of two of these promoters were reported previously by Talkington & Pero, 1979). All five promoters shared highly conserved nucleotide sequences that were centered at about 35 and 10 base-pairs upstream from their start points of transcription. Based on these conserved sequences we propose that RNA polymerase containing gp28 recognizes the prototype sequence 5′T- -T-AGGAGA- -A-TT in the −35 promoter region and the sequence 5′TTT-TTT in the −10 region. (In SP01 DNA, T is the thymine analog 5-hydroxymethyluracil.) These prototype sequences differ strikingly from the corresponding conserved regions of SP01 early gene promoters which are recognized by the unmodified B. subtilis RNA polymerase and which are highly homologous to promoters for Excherichia coli RNA polymerase. These findings suggest that sigma factors (host sigma and gp28) dictate the recognition of both the −35 and −10 regions of promoters.

Polycystic ovarian condition in the dehydroepiandrosterone‐treated rat model: Hyperandrogenism and the resumption of meiosis are major initial events associated with cystogenesis of antral follicles

The purpose of this study was to elucidate the early effects of dehydroepiandrosterone (DHEA) in the polycystic rat model by charting cytological changes in the early antral follicle of the ovary and constructing a serum hormonal profile. Histological examinations of ovaries from DHEA‐treated rats for ten consecutive days revealed that the oocyte of antral follicles, ranging from 1.5 mm to 3.4 mm in diameter, had become activated, i.e., had resumed meiosis. Tabulation and statistical analysis revealed a highly significant difference in the percentage of oocyte activation between the ovaries of DHEA‐treated and control rats. Granulosa cells associated with those antral follicles included in our statistical analysis showed no evidence of atresia. A few follicles not included in our analysis contained oocytes that had resumed meiosis and whose associated granulosa cells were atretic. The observed resumption of meiosis occurred in the absence of surges of follicle stimulating hormone (FSH) and luteinizing hormone (LH). During meiosis, a period when many oocytes become activated, levels of serum androgens (DHEA, testosterone, and androstenedione) were high, while FSH, LH, and prolactin (PRL) levels did not differ significantly from those in the controls. Follicles that resume meiosis may be members of a group of follicles that produces a signal(s) when the oocyte becomes uncoupled from the granulosa cell. This signal(s) permit(s) a reprogramming of the accompanying granulosa cells of the follicle to engage in certain developmental processes of cystogenesis. Just what cascade of signals is necessary to achieve this selection remains elusive at this time and is the subject of our continuing investigations. Anat. Rec. 249:44–53, 1997.

The participation of growth factors in simulating the quiescent, proliferate, and differentiate stages of rat granulosa cells grown in a serum-free medium

Ovarian granulosa cells from small antral follicles from immature rats were cultured in a serum-free medium on an extracellular matrix for 10 days with growth factors in an effort to simulate the metabolic states they experience during their differentiation. During in vivo differentiation granulosa cells are initially quiescent, later proliferate and subsequently commence differentiation. With the production of androstenedione by the vascularized theca interna they produce estrogen and when the follicle reaches the preovulatory stage, granulosa cells produce both estrogen and progesterone. Culturing granulosa cells in serum-free medium plus FSH, PDGF, or FSH plus PDGF, the cells remain quiescent. The cells proliferate most consistently (as assessed by DNA quantitation) when cultured in FSH, PDGF, TGF alpha, TGF beta and GH, and undergo the first level of differentiation by producing estrogen (assessed by RIA) when cultured in FSH, PDGF, TGF beta, IGF-I and delta 4-A. Further differentiation is achieved in the presence of FSH, PDGF, TGF alpha, bFGF and delta 4-A when the cells produce both estrogen and progesterone similar to their production in preovulatory follicles. Phase contrast photomicrographs were made to monitor cellular shape changes. Electron microscopic analysis of the quiescent and proliferative cells reveal them to contain the normally occurring organelles. After 8 days in culture, cells producing estrogen, and estrogen and progesterone, contain endoplasmic reticulum of the smooth variety, an organelle which, in cooperation with mitochondria, is known to be involved in the production of steroids such as estrogen and progesterone. Therefore, with the addition of one or more growth factors and androstenedione to FSH-containing serum free medium, the simulated conditions are partially reminiscent of the follicular microenvironment, in which granulosa cells cultured on extracellular matrix can exhibit characteristics of growth and differentiation similar to folliculogenesis.

Transcription of cloned DNA from Bacillus subtilis phage SP01 requirement for hydroxymethyluracil-containing DNA by phage-modified RNA Polymerase☆

Abstract We have cloned endonuclease restriction fragments of Bacillus subtilis phage SP01 DNA in a phage lambda vector, thereby replacing 5-hydroxymethyluracil (the thymine analog in SP01 DNA) with thymine. Two cloned fragments were shown to contain phage SP01 “early” genes. These cloned DNAs, as well as the 5-hydroxymethyluracil-containing fragments from which they were derived, supported specific RNA synthesis by B. subtilis RNA polymerase. Two other cloned DNAs contained phage “middle” genes, sequences that are known to be transcribed by a modified form of B. subtilis RNA polymerase containing a phage-coded regulatory protein (gp28) in place of the host sigma factor. The cloned middle genes failed to support specific RNA synthesis by phage-modified polymerase although the corresponding 5-hydroxymethyluracil-containing fragments were effective templates for in vitro transcription. This difference in the template activity of cloned and native SP01 fragments could not be attributed to promoter “mutations” introduced during replication in Escherichia coli as the nucleotide sequence of a middle gene promoter was identical for both thymine and 5-hydroxymethyluracil-containing DNA. We therefore conclude that, at least under certain conditions of in vitro RNA synthesis, 5-hydroxymethyluracil is required for specific transcription by gp28-containing RNA polymerase.