Structure of the goat psi beta y beta-globin pseudogene. Analysis of goat pseudogene evolutionary patterns

The 12-member beta-globin gene locus of the goat contains three beta(adult)-type pseudogenes, one in each of three four-gene subsets of the locus. We have determined the complete nucleotide sequence of psi beta y, the pseudogene present in the most downstream four-gene subset, which also contains the functional fetal gene, beta F. psi beta y contains, throughout its length, numerous incapacitating mutations in common with the previously sequenced goat psi beta x and psi beta z pseudogenes consistent with the model that all were descended from a common pseudogene ancestor which became defective prior to the expansion of the beta-globin locus in the goat lineage. Evolutionary analysis of the psi beta y sequence in comparison to psi beta x and psi beta z provides evidence that nucleotide substitutions were fixed in a random manner within these pseudogenes with respect to polarity, coding versus non-coding regions, and replacement sites versus silent sites. However, substitutions appear to have accumulated asymmetrically between different pseudogenes in a manner that provides evidence for partial gene conversion. Moreover, the presence of deletions in goat psi beta y, which are also observed in the cow pseudogene psi 2, but not in the cow psi 1 pseudogene, indicate that goat psi beta y and cow psi 2 are orthologous but cow psi 1 actually arose prior to the goat/cow divergence. The authentic goat orthologue to cow psi 1 temporarily existed in the goat lineage but was deleted, probably prior to the divergence of goats and sheep.     

The heme environment of ovoperoxidase as determined by optical spectroscopy

Native ovoperoxidase exhibited an optical absorption spectrum with certain similarities to lactoperoxidase, but not horseradish peroxidase, over the pH range 4.5-11.5. Ovoperoxidase had three distinct spectral forms dependent on pH, with transitions at apparent pKa values of 6.6 and 3.0. Complexes of ovoperoxidase with CN-, N3-, F-, or when reduced and ligated to carbon monoxide, CN-, or pyridine, were distinct from other peroxidases. Ovoperoxidase formed two specific and different spectral derivatives at pH 6.0 and 8.0, either in the native state, or when combined with CN-, when reduced, or when reduced and ligated to CN-. The position of the Soret band when mixed with near-stoichiometric amounts of H2O2. This cycling was inhibited by phenylhydrazine, 3-amino-1,2,4-triazole, or low pH (less than or equal to 6). Compound II was formed when ovoperoxidase was mixed with ethyl hydrogen peroxide in a 1:3 ratio, but not with H2O2. With a great excess of H2O2, Compound III was formed at pH 8.0; at pH 6.0 or below, the Soret band shifted slightly with excess of H2O2, but Compound III was never formed. Even when ovoperoxidase was bound to proteoliaisin (Weidman, P. J., and Shapiro, B. M. (1987) J. Cell Biol. 105, 561-567), ovoperoxidase exhibited spectral characteristics of the free enzyme.     

Identification of functional arginines in human angiogenin by site-directed mutagenesis.

Chemical modifications of human angiogenin had suggested that arginines are essential for its ribonucleolytic activity [Shapiro, R., Weremowicz, S., Riordan, J. F., & Vallee, B. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8783-8787]. Each of the six arginines within or near angiogenin's catalytic or cell-binding sites--i.e., those at positions 5, 31, 32, 33, 66, and 70--was therefore mutated to alanine. Two of these residues, Arg-5 and Arg-33, indeed play a role, albeit noncrucial, in enzymatic activity, although neither one is implicated in the abolition of activity by arginine reagents. R5A-angiogenin, while nearly fully active toward dinucleotides, is one-fourth as active as angiogenin toward tRNA, suggesting that Arg-5 may participate in the binding of peripheral components of the substrate. In contrast, the activity of R33A-angiogenin toward both polynucleotide and dinucleotide substrates is reduced similarly, reflecting a decrease in kcat. These results, together with its position in the calculated three-dimensional structure of angiogenin, imply an indirect role for Arg-33 in catalysis. Three arginines are important for angiogenesis: mutation of Arg-5, Arg-33, or Arg-66 dramatically reduces the angiogenic potency of angiogenin on the chicken embryo chorioallantoic membrane. Arg-66 lies within a segment previously proposed to be part of a cell-surface receptor binding site. Arg-5 and Arg-33 are outside of this site as defined at present, and the decreased angiogenicity of R5A- and R33A-angiogenin may be a consequence of their reduced ribonucleolytic activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Logarithmic amplifiers

Logarithmic amplifiers (log amps), which produce an output signal proportional to the logarithm of the input signal, are widely used in cytometry for measurements of parameters that vary over a wide dynamic range, e.g., cell surface immunofluorescence. Existing log amp circuits all deviate to some extent from ideal performance with respect to dynamic range and fidelity to the logarithmic curve; accuracy in quantitative analysis using log amps therefore requires that log amps be individually calibrated. However, accuracy and precision may be limited by photon statistics and system noise when very low level input signals are encountered.     

Effect of an acute bout of exercise on glucose disposal in human obesity

The effect of acute exercise on insulin action has been studied in six obese (150-250% ideal body weight) non-insulin-dependent diabetics (OD), seven obese normoglycemics (ON), and six lean healthy controls (LC). Using a three-stage euglycemic clamp, the metabolic clearance rate (MCR) of glucose under increasing insulin concentrations was measured. The insulin dose-response curve was assessed on two separate occasions: 1) a base-line test and 2) 1 h after aerobic treadmill exercise at a steady-state heart rate of 150-160 beats/min. In the base-line test, under all insulin levels, glucose MCR was significantly lower in obese compared with lean individuals (P less than 0.01). Exercise increased glucose MCR at the highest hormonal concentrations applied to 124 and 134% of base line in OD and in ON, respectively (P less than 0.05); the insulin concentration required for one-half of the maximal clearance rate of glucose achieved in this study decreased from 200 to 130 and from 160 to 95 microU/ml in OD and ON, respectively (P less than 0.05). The changes in these parameters were insignificant in LC. It is suggested that acute exercise affected the insulin dose-response curve in OD and in ON but not in LC; although enhanced by exercise, glucose MCR remained significantly lower in both obese groups compared with control subjects. We concluded that insulin resistance, which accompanies extreme obesity, could be markedly decreased but not completely reversed by one bout of exercise.     

Evolutionary analysis of the multigene pregnancy-specific β1-Glycoprotein family: Separation of historical and nonhistorical signals

The pregnancy-specific ₁-glycoproteins (PSG) form a large family of closely related proteins. Using newly developed methods of sequence analysis, in combination with protein modeling, we provide a framework for investigating the evolution and biological function of genes like the PSG. Evolutionary trees, based on C-terminal sequence, group PSG genes in a manner consistent with their genomic organization. Trees constructed using the N-terminal domain sequences are unreliable as an indicator of phylogeny because of non-neutral processes of sequence change. During duplication of the PSG genes, evolutionary pressures have resulted in a gradient of constrained change across each gene. The N-terminal domains show a nonrandom pattern of amino acid substitutions clustered in the immunoglobulin complementarity-determining region (CDR)-like regions, which appear to be important in the function of the protein.     

Pseudohypoparathyroidism type Ia: two new heterozygous frameshift mutations in exons 5 and 10 of the Gsα gene

Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disease characterized by resistance to PTH and other hormones that act via cAMP. Patients have deficient activity of Gs, the subunit of the G protein, which couples hormone receptors to stimulation of adenylate cyclase. We describe two new mutations discovered in two sporadic patients with PHP-Ia. Using genomic DNA, we have amplified exons 2–13 of the Gs gene (GNAS1) by PCR, and sequenced the resulting products. Both patients had Albright's hereditary osteodystrophy, resistance to multiple hormones, and deficient Gs activity. In the first patient, a deletion of a C in exon 5 at codon 115 was found. In the second patient, an insertion of a C in exon 10 at codon 267 was detected. Both these heterozygous mutations cause frameshift, and predict decreased production of Gs. This report adds two new Gs mutations to the known ten mutations recently described.     

Biotype-specific expression of dsRNA in the sweetpotato whitefly

This study was conducted to evaluate the effect of two different biotypes of the sweetpotato whitefly,Bemisia tabaci (Gennadius), on the induction of squash silverleaf (SSL), and to determine if double-stranded RNA (dsRNA) occurs in geographically remote populations of the two biotypes. Recently collected B-biotype whiteflies from Florida, Arizona, Mississippi, and Texas (SPW-B) all contained a 7.0 kb dsRNA molecule. Kb dsRNA molecule. Laboratory colonies of A-biotype whiteflies that were originally collected in 1981 from cotton in Arizona and California did not contain the 7.0 Kb dsRNA. When the two biotypes were compared only the SPW-B induced rapid onset, grade 5, SSL. DsRNA similar to that found in adult SPW-B was concentrated in whitefly nymphs, but host plant leaf tissue did not contain any consistent dsRNA molecules. SPW-A only induced low-grade SSL and progeny of SPW-A that were fed on pumpkin plants displaying SSL did not acquire the ability to express dsRNA or induce SSL. Our data suggest that dsRNA is not directly involved in the induction of SSL and that SSL is a host-specific response, to a feeding injury induced by B-biotype whiteflies. The origin and source of the 7.0 Kb dsRNA molecule remains enigmatic but its expression is constant in the whitefly biotype that is responsible for the induction of SSL and several other plant disorders in the U.S.     

The roles of starvation and selective substrates in the emergence of araB-lacZ fusion clones.

The araB-lacZ fusion system has been a key case in the 'directed mutation' controversy. Fusions did not occur detectably during normal growth but formed readily after prolonged incubation on selective Ara-Lac medium. To distinguish the roles of starvation stress and selective substrates in coding sequence fusions, we applied sib selection and PCR technologies. Sib selection of the prefusion strain, MCS2, starved under aerobic conditions permitted us to isolate active fusion clones which had never been in contact with arabinose or lactose. Hence, a directive role for selective substrates is not essential. Aerobiosis was necessary for fusions to appear in glucose-starved cultures. The difference in fusion formation between normal and starved conditions is best explained by the response of a signal transduction network to physiological stimuli to activate Mu prophage joining of araB and lacZ sequences. PCR analysis revealed that direct plating on selective Ara-Lac agar yielded mostly a single class of 'standard' fusions, while sib selection yielded a broader spectrum of fusion structures. Standard fusions were found to occur within a narrow 9 bp window in lacZ. The high frequency of standard fusions in glucose-starved cultures suggested efficient and/or specific Mu action.     

Application of 3-Dimensional Homology Modeling of Cytochrome P450 2B1 for Interpretation of Site-Directed Mutagenesis Results

Abstract Three-dimensional structures of cytochrome P450 2B1 were modeled based on the crystallographic structure of P450(cam). The effect of the alignment, loop choice, and minimization with or without water was assessed. Although final models were similar in overall structure, the identity of active site residues depended upon the alignment. An example is Phe-206, which may or may not form part of the active site. The choice of the loop conformation had a lesser effect, while including water in the final minimization step was essential for preserving the shape and size of the active site. The best model (model 2) was in good agreement with the data from site-directed mutagenesis studies, and correctly predicted the effect of substitutions at 9 out of 10 amino acid positions. Thus, residues important for P450 2B1 activity, such as Ile- 114, Phe-206, Ile-290, Thr-302, Val-363, and Gly-478, constitute part of the active site and are able to interact with the substrate androstenedione through hydrophobic interactions. On the other hand, Ser-303, Ser-360 and Lys-473 are far from the active site and/or cannot interact with the substrate, in agreement with experimental data. The model indicates other residues likely to be important for enzyme function, such as Tyr- 111, Leu-209, Ile-477, and Ile- 480, which can be tested experimentally. The substrate may assume numerous binding orientations consistent with observed patterns of hydroxylation at C(5) and C(6). The replacement in the model of certain amino acid residues to mimic residue substitutions from site-directed mutagenesis studies and docking of the substrate into the modified active site allowed a plausible explanation for alterations in regio- and stereospecificities of some mutants of P450 2B1, such as Gly-478 → Ala or Val-363 Ala.