Mutations in the mitochondrial split gene COXI are preferentially located in exons: a mapping study of 170 mutants

We have analysed the precise location of a large number (170) of mutations affecting the structural gene for subunit 1 of the cytochrome c oxidase complex. This gene, COXI, is 12.9 kb long and the major part of the sequence (i.e. 11.3 kb) is composed of introns. Several conclusions can be drawn from this study: (1) A significant proportion (84/170) of the mutations cannot be assigned to a single position within the gene by deletion mapping, in spite of clearly being located in it. These mutations are probably large deletions or multiple mutations. (2) Four mutants carry distant double mutations, which have been individually localized. (3) Eighty-two mutants have lesions that are restricted to very short regions of the gene and we therefore conclude that they are most probably due to single hits; amongst these single mutations, 41 are unambiguously located in exons and 28 in introns. This result implies that, at least in this particular split gene, the probability of selection of a mutant phenotype in an exon is, on the average, 13.3 times greater than in an intron, in spite of the existence, within most of these introns, of open reading frames specifying intronic proteins. The evolutionary significance and biological implications of these results are discussed.     

Effects of anatomic variability on blood flow and pressure gradients in the pulmonary capillaries

Dhadwal, Amit, Barry Wiggs, Claire M. Doerschuk, and RogerD. Kamm. Effects of anatomic variability on blood flow and pressure gradients in the pulmonary capillaries. J. Appl. Physiol. 83(5): 1711-1720, 1997.Atheoretical model is developed to simulate the flow of blood throughthe capillary network in a single alveolar septum. The objective is tostudy the influence of random variability in capillary dimension andcompliance on flow patterns and pressures within the network. Thecapillary bed is represented as an interconnected rectangular grid ofcapillary segments and junctions; blood flow is produced by applying apressure gradient across the network. Preferred flow channels are shownto be a natural consequence of random anatomic variability, the effectof which is accentuated at low transcapillary pressures. Thedistribution of pressure drops across single capillary segments widenswith increasing network variability and decreasing capillary transmuralpressure. Blockage of one capillary segment causes the pressure dropacross that segment to increase by 60%, but the increase falls to<10% at a distance of three segments. The factors that causenonuniform capillary blood flow through the capillary network arediscussed.

     

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

Abstract An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae , and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes , and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.     

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.     

Peroxidases in Acetabularia: their possible role in development

Abstract. Crude enzymatic extracts from Acetabularia exhibit very low peroxidase activity after a lag period. Starch gel electrophoresis of extracts from growing algae shows a single, extremely anodic band. Extracts of small, slow-growing or cap-bearing algae, which do not grow any more, do not exhibit any peroxidase band. Cytochemical staining with benzidine reveals changes in both the quantity and distribution of peroxidase along the polarized Acetabularia cell. The homogenous staining of small algae becomes distributed along a negative apico-basal gradient when the algae initiate their rapid growth phase. This polarized pattern is repeated on the hair whorls. A similar developmental sequence directs cap growth, with an initial intense staining reaction of the primordium, which later leaves only the corona inferior stained blue. Finally, the Acetabularia cell remains slightly blue at the edges of the rhizoidal out-growths and cap rays. Crude extracts of Acetabularia induce a lag in standard horseradish peroxidase (HRP) activity. The inhibitor is always present in small and growing algae; it is sometimes absent or less active in cap-bearing algae. In no case does it change the kinetics of the HRP reaction with guaïacol. The lag is completely suppressed by pretreatment with either H₂O₂ or ascorbate oxidase. The changes in peroxidase activity, correlated with developmental stage and according to a polarized gradient, suggest that the enzyme could be involved in some way in the control of morphogenesis in Acetabularia . An inhibitor of peroxidase activity, which disappears as the cap matures, might, in turn, exert a regulatory function.     

Del11p13/nephroblastoma without aniridia

Summary A patient is reported with del11p13, low catalase level, nephroblastoma, chordee and cryptorchidism, no evident mental retardation, and with normal irides. This unique observation suggests the following order of loci in 11p13, from centromere to telomere: catalase, Wilms tumor, aniridia. The chromosomal origin of nephroblastoma may be more frequent than estimated on the basis of its association with aniridia.     

Substrate positions and induced-fit in crystalline adenylate kinase.

The binding positions of ATP and AMP in pig muscle adenylate kinase (EC 2.7.4.3) have been located by X-ray diffraction analysis. For this purpose crystals have been soaked with solutions containing substrates and substrate analogues. Two adenosine pockets and the region of the phosphates have been identified. In combination with other experimental data the pockets have been assigned to the AMP site and the ATP site, respectively. Moreover, the results suggest that the known conformations of adenylate kinase reflect an induced-fit of the enzyme: conformation B being related to the free enzyme E and conformation A being related to E¹, the enzyme species after a substrate-induced conformational change.     

Proline excretion in Escherichia coli: A comparison of an argD + strain and a proline-excreting argD − derivative

In an attempt to deduce the physiological basis of proline excretion in argD ^– strains of Escherichia coli K12, several properties of an argD ⁺ (nonexcreting) and an argD ^– (excreting) derivative were compared. No difference was found in the transport or in the utilization of either proline or its immediate precursor, ¹-pyrroline-5-carboxylate (PCA). Furthermore, no differences were found in the physical or kinetic properties of partially purified preparations of the enzyme mediating the final step in proline biosynthesis, PCA reductase. The specific activity of PCA reductase was, however, consistently higher in crude extracts prepared from the argD ^– mutant.This work was supported by grants from the National Institutes of Allergy and Infectious Diseases (Public Health Service No. AI-10862) and The University of Connecticut Research Foundation (to C. M. B.). J. J. R. was supported by an NDEA Predoctoral Fellowship.