Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′.     

Fetal sheep adrenal blood flow responses to hypoxemia after splanchnicotomy using fluorescent microspheres

Buchwalder, Lynn F., Michelle Lin, Thomas J. McDonald, andPeter W. Nathanielsz. Fetal sheep adrenal blood flow responses tohypoxemia after splanchnicotomy using fluorescent microspheres. J. Appl. Physiol. 84(1): 82-89, 1998.Adrenal gland blood flow (ABF) increases during hypoxemia infetal sheep, but regulation of ABF is poorly understood. The purpose ofthis study was to determine the effects of splanchnic nerve section onfetal ABF responses to hypoxemia using the fluorescent microsphere (FM) technique. At 125 days of gestation, 14 unanesthetized fetal sheep [bilateral splanchnicotomy (Splx,n = 6) and control (Cont,n = 8)] were injectedwith FM before and at 60 min ofN₂-induced hypoxemia (~40%decrease in fetal arterial PO₂).Adrenal tissue and reference blood samples were digested and filtered, and FM dye was extracted for spectrometer analysis. Baseline whole, medullary, and cortical ABF for the Cont group were similar to published values using radioactive microspheres and did not differ fromSplx values. Hypoxemia increased whole, medullary, and cortical ABF(mean ± SE) from baseline for the Cont group by 281 ± 35, 258 ± 31, and 496 ± 81% (P < 0.05). The increase for the Splx group was attenuated compared with theCont group (P < 0.05) for whole andmedullary ABF (139 ± 27 and 43 ± 27%) but not cortical ABF(326 ± 91%). We conclude that1) the FM technique is valid formeasuring fetal ABF and 2) in fetalsheep the splanchnic nerve is not necessary to maintain basal ABF butplays an important role in regulating the hypoxemia-induced increase inABF through the medullary, but not cortical, ABF response.

     

Effect of air breathing on acid-base and ion regulation after exhaustive exercise and during low pH exposure in the bowfin, Amia calva.

To explore a potential conflict between air breathing and acid-base regulation in the bowfin (Amia calva), we examined how individuals with access to air differed from fish without air access in their response to acidosis. After exhaustive exercise, bowfin with access to air recovered significantly more slowly from the acidosis than fish without air access. While arterial blood pH (pH(a)) of fish without air access recovered to resting levels by 8 h, pH(a) was still significantly depressed in fish having access to air. In addition, Pco(2) was slightly more elevated in fish having air access than those without it. Fish with access to air still had a significant metabolic acid load after 8-h recovery, while those without air access completely cleared the load within 4 h. These results suggest that bowfin with access to air were breathing air and, consequently, were less able to excrete CO(2) and H(+) and experienced a delayed recovery. In contrast, during exposure to low pH, air breathing seemed to have a protective effect on acid-base status in bowfin. During exposure to low pH water, bowfin with access to air developed a much milder acidosis than bowfin without air access. The more severe acidosis in fish without air access was caused by an increased rate of lactic acid production. It appears that enhanced O(2) delivery allowed air-breathing bowfin to avoid acidosis-induced anaerobic metabolism and lactic acid production. In addition, during low pH exposure, plasma Na(+) and Cl(-) concentrations of fish without air access fell slightly more rapidly than those in fish with air access, indicating that the branchial ventilatory changes associated with air breathing limited, to some degree, ion losses associated with low pH exposure.     

Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields active S phase cyclin-Cdk.

Selective degradation of single subunits of multimeric complexes by the ubiquitin pathway underlies multiple regulatory switches, including those involving cyclins and Cdk inhibitors. The machinery that segregates ubiquitinated proteins from unmodified partners prior to degradation remains undefined. We report that ubiquitinated Sic1 (Ub-Sic1) embedded within inactive S phase cyclin-Cdk (S-Cdk) complexes was rapidly degraded by purified 26S proteasomes, yielding active S-Cdk. Mutant proteasomes that failed to degrade Ub-Sic1 activated S-Cdk only partially in an ATP-dependent manner. Whereas Ub-Sic1 was degraded within approximately 2 min, spontaneous dissociation of Ub-Sic1 from S-Cdk was approximately 200-fold slower. We propose that the 26S proteasome has the intrinsic capability to extract, unfold, and degrade ubiquitinated proteins while releasing bound partners untouched. Activation of S-Cdk reported herein represents a complete reconstitution of the regulatory switch underlying the G1/S transition in budding yeast.     

Dependence of amino acid composition upon nitrogen availability in birch (Betula pendula)

Small birch plants ( Betula pendula Roth .) were grown at different rates of exponentially increasing nitrogen supply. This resulted in plants with different relative growth rates and different internal nitrogen concentrations. Within a nitrogen treatment, both of these variables remained constant with time.
Free amino acids were measured in leaves and roots of the seedlings at two different harvests. At greater nitrogen supply, higher concentrations of total amino acid nitrogen were found in roots and leaves. The ratio of amino acid nitrogen to total nitrogen was low albeit greater at higher nitrogen supply. Higher concentrations of amino acid nitrogen were mainly due to high concentrations of citrulline, glutamine, γ-aminobuitric acid and arginine.
Greater leaf concentrations of amino acid nitrogen at higher nitrogen supply may be related lo increased concentrations in the xylem sap and/or may be indicative of small excesses of nitrogen with respect to current nitrogen usage in protein synthesis.     

Detection of Met-enkephalin and Leu-enkephalin in the posterior pituitary of the holostean fish, Amia calva

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 (n = 4). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8) and dynorphin B(1-13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.     

Effects of season and testosterone treatment on gonadotrophin secretion and pituitary responsiveness to gonadotrophin-releasing hormone in castrated Romney and Poll Dorset rams.

In castrated rams (Romney and Poll Dorset, n = 8 for each breed), inhibition by testosterone treatment (administered via Silastic capsules) of luteinizing hormone (LH) pulse frequency, basal and mean LH concentrations, mean follicle-stimulating hormone (FSH) concentration, and the peak and total LH responses to exogenous gonadotrophin-releasing hormone (GnRH) were significantly (P less than 0.01) greater during the nonbreeding than during the breeding season. Poll Dorset rams were less sensitive to testosterone treatment than Romney rams. In rams not receiving testosterone treatment, LH pulse frequency was significantly (P less than 0.05) lower during the nonbreeding season than during the breeding season in the Romneys (15.8 +/- 0.9 versus 12.0 +/- 0.4 pulses in 8 h), but not in the Poll Dorsets (13.6 +/- 1.2 versus 12.8 +/- 0.8 pulses in 8 h). It is concluded that, in rams, season influences gonadotrophin secretion through a steroid-independent effect (directly on hypothalamic GnRH secretion) and a steroid-dependent effect (indirectly on the sensitivity of the hypothalamo-pituitary axis to the negative feedback of testosterone). The magnitude of these effects appears to be related to the seasonality of the breed.     

Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys

Using the strictly neutral model as a null hypothesis, we tested for deviations from expected levels of nucleotide polymorphism at the alcohol dehydrogenase locus (Adh-1) within and among four species of pocket gophers (Geomys bursarius major, G. knoxjonesi, G. texensis llanensis, and G. attwateri). The complete protein-encoding region was examined, and 10 unique alleles, representing both electromorphic and cryptic alleles, were used to test hypotheses (e.g., the neutral model) concerning the maintenance of genetic variation. Nineteen variable sites were identified among the 10 alleles examined, including 9 segregating sites occurring in synonymous positions and 10 that were nonsynonymous. Several statistical methods, including those that test for within-species variation as well as those that examine variation within and among species, failed to reject the null hypothesis that variation (both within and between species of Geomys) at the Adh locus is consistent with the neutral theory. However, there was significant heterogeneity in the ratio of polymorphism to divergence across the gene, with polymorphisms clustered in the first half of the coding region and fixed differences clustered in the second half of the gene. Two alternative hypotheses are discussed as possible explanations for this heterogeneity: an old balanced polymorphism in the first half of the gene or a recent selective sweep in the second half of the gene.      

The evolutionary analysis of the Tnt1 retrotransposon in Nicotiana species reveals the high variability of its regulatory sequences

We studied the evolution of the tobacco Tnt1 retrotransposon by analyzing Tnt1 partial sequences containing both coding domains and U3 regulatory sequences obtained from a number of Nicotiana species. We detected three different subfamilies of Tnt1 elements, Tnt1A, Tnt1B, and Tnt1C, that differ completely in their U3 regions but share conserved flanking coding and LTR regions. U3 divergence between the three subfamilies is found in the region that contains the regulatory sequences that control the expression of the well-characterized Tnt1-94 element. This suggests that expression of the three Tnt1 subfamilies might be differently regulated. The three Tnt1 subfamilies were present in the Nicotiana genome at the time of species divergence, but have evolved independently since then in the different genomes. Each Tnt1 subfamily seems to have conserved its ability to transpose in a limited and different number of Nicotiana species. Our results illustrate the high variability of Tnt1 regulatory sequences. We propose that this high sequence variability could allow these elements to evolve regulatory mechanisms in order to optimize their coexistence with their host genome.      

Potent piperazine hydroxyethylamine HIV protease inhibitors containing novel P3 ligands

The 2-isopropyl thiazolyl group is a highly optimized P₃ ligand for C₂ symmetry-based HIV protease inhibitors, as exemplified in the drug ritonavir. Here we report that incorporation of this P₃ ligand into a piperazine hydroxyethylamine series also yielded novel, highly potent inhibitors. In tissue culture assays, the presence of human serum was less deleterious to the activity of these inhibitors than to that of ritonavir. Furthermore, potent activity against ritonavir resistant HIV was observed.