Biomedical subjects
Publications and source records attributed to K McEntee.
Pheochromocytomas and ultimobranchial (C-cell) neoplasms were diagnosed at a high frequency in a family of Guernsey bulls. The incidence supported the hypothesis of autosomal dominant inheritance of the neoplasms. These two neoplasms also occur in man as multiple endocrine adenomatosis type 2, which has autosomal dominant inheritance.
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Cultures of Mycoplasma bovis were injected into the vesicular glands of bulls in order to determine the pathogenicity of M. bovis in the vesicular gland. In addition M. bovis was administered to bulls by intravenous and intra-articular injection in order to mimic possible natural routes of infection. Direct intravesicular injection of M. bovis resulted in seminal vesiculitis with persistant shedding of mycoplasma in the vesicular gland secretion for up to 81/2 months. Attempts to reproduce vesiculitis by other routes were not successful.
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The DNA-dependent ATPase activity of the recA protein of Escherichia coli shows a complex dependence on ATP concentration. With a single-stranded (SS) DNA cofactor, the Hill coefficient for ATP is 3.3 at pH 8.1 and 1.4 at pH 6.2. With a double-stranded (DS) DNA cofactor, the Hill coefficient is 3.3 at pH 6.2 (no reaction is detectable at pH 8.1). In the presence of SS DNA, the Km for ATP is 20 microM, independent of pH, while with DS DNA at pH 6.2, KmATP is 100 microM. These and other observations indicate that the interaction of recA protein with ATP is influenced by both pH and DNA cofactor. ADP, UTP, dTTP, and GTP are competitive inhibitors of the ATPase activity of recA protein, indicating that there is a single binding site for nucleoside triphosphates. Nucleoside triphosphates, but not ADP, reduce the Hill coefficient for ATP hydrolysis and thus can contribute to the cooperative effect of ATP.
Incubation of the recA protein of Escherichia coli with the ATP analog adenosine 5'-O-(3-thiotriphosphate) (ATP(gamma S)) in the presence of DNA produces an irreversible inhibition of ATPase activity, although in the presence of ATP, ATP(gamma S) shows an initial competitive inhibition. ATP(gamma S) is not appreciably hydrolyzed by recA protein and the inhibition of ATPase activity is due to the formation of stable complexes which contain equimolar amounts of ATP(gamma S) and recA protein. Formation of stable complexes requires DNA, which is also stably bound to recA protein in the presence of ATP(gammaS), at a ratio of 5 to 10 nucleotides/recA protein monomer. The DNA requirement is satisfied by either single-or double-stranded DNA, and in the latter case, the pH dependence is comparable to that observed for ATP hydrolysis. Binding of ATP(gamma S) is inhibited by other nucleoside di- and triphosphates with efficiencies corresponding to their inhibitory effects on the ATPase activity of recA protein.
Both single- and double-stranded DNA stimulate the hydrolysis of ATP catalyzed by the recA protein of Escherichia coli. However, the reactions differ in their pH optima, response to recA protein concentration, salt sensitivity, and degree of inhibition by ADP, all of which reflect different requirements for the prehydrolytic binding of single- and double-stranded DNA by the RecA protein. Single- and double-stranded DNA stimulate hydrolysis of the same nucleoside triphosphates, principally (r,d)ATP and (r,d)UTP, suggesting that a single hydrolytic site is utilized in both single- and double-stranded DNA-dependent reactions. recA protein also catalyzes detectable ATP hydrolysis in the absence of exogenous DNA, although the rate is reduced 2 to 3 orders of magnitude. This DNA-independent hydrolysis shows the same nucleotide specificity at pH 6.2 and 7.5, although the rate of hydrolysis depends upon the pH.
The recA protein of Escherichia coli binds both single- (SS) and double-stranded (DS) DNA; however, the optimal conditions differ for interaction with these DNA substrates. Binding of DS DNA by recA protein is pH dependent (optimum near pH 6.2) and requires a nucleoside triphosphate (ATP) and divalent cation. Substitution of the 5'-O-3'-thiotriphosphate (ATP(gamma S)) for ATP leads to formation of stable complexes of recA protein and DNA that dissociate very slowly. Formation of these complexes is extremely sensitive to ionic strength and pH. However, once formed, the complexes resist changes in pH and high salt concentrations. SS DNA binds to recA protein in the absence of a nucleoside triphosphate, but recA protein-SS DNA complexes are stabilized by ATP(gamma S). At high recA protein/DNA ratios (1 recA protein monomer/30 nucleotides), these complexes sediment in sucrose gradients as large protein-DNA aggregates. Although ATP(gamma S) blocks dissociation of recA protein from DNA, ATP stimulates the release of recA protein from SS DNA. Hydrolysis of the ATP is not required for dissociation since it is also enhanced by ADP and certain nucleoside triphosphates that are not hydrolyzed by recA protein. recA protein binds with different affinities to ribohomopolymers and deoxyhomopolymers. It preferentially binds polydeoxythymidylate and polydeoxycytidylate but does not bind short oligonucleotides, indicating that there is a minimum size requirement for the binding step. The recA protein exists as a heterogeneous aggregate at pH 7.5 and at low ionic strength. At pH 6.2 in the presence of Mg2+, the protein sediments homogeneously as a dimer. At pH 6.2, ATP or ATP(gamma S) promotes an oligomerization of the recA protein which can be observed as filamentous structures by electron microscopy. Oligomerization is not induced by UTP, a nucleoside triphosphate that is efficiently hydrolyzed by the recA protein, but fails to stimulate efficiently recA protein-promoted annealing and assimilation of single-stranded DNA.
Hydrolysis of UTP catalyzed by the recA protein of Escherichia coli is stimulated by both double- (DS) and single-stranded (SS) DNA. DS DNA-dependent UTPase activity has a sharp optimum near pH 6. SS DNA-dependent UTP hydrolysis also is optimal near pH 6, although considerable activity remains at pH 8. Both SS and DS DNA-dependent UTPase activities are nonlinearly dependent on recA protein concentration at pH 6 but the SS DNA-dependent reaction shows a linear dependence on enzyme concentration at pH 8. The Km for UTP in the SS DNA-dependent reaction decreases from 147 microM at pH 8 to 33 microM at pH 6. The Km for UTP in the DS DNA-dependent reaction is 247 microM at pH 6. In addition, the Hill coefficient for UTP in the SS DNA-dependent reaction decreases from 3.5 at pH 8 to 1.9 at pH 6, while in the DS DNA-dependent reaction, the Hill coefficient is 2.4 at pH 6. Thus, the UTPase activity of the recA protein differs from the ATPase activity of recA protein primarily in the pH dependence of KmUTP, Vmax, and response to enzyme concentration of the SS DNA-dependent hydrolysis reaction. These differences may be related to the inability of UTP to substitute effectively for ATP in recA protein-promoted annealing and assimilation of SS DNA.
A simple and rapid three-step procedure for the large scale purification of the recA protein of Escherichia coli is described. The method depends primarily on a single chromatographic step which is highly specific for recA protein: elution by ATP from single-stranded DNA cellulose. With this procedure, gram quantities of recA protein, greater than 99% pure, can be reproducibility prepared for biochemical and biophysical analysis.
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The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains.
The International Registry of Reproductive Pathology contains more than 19,000 case records. It is indexed with the aid of Systematized Nomenclature of Medicine (SNOMED) codes and the MUMPS-11 interactive computer language. A package of programs was developed to maintain an index file of cases in the collection and to produce for each species a printed list of case numbers for every combination of topography, morphology and etiology. The printed disease data from the computer are in English with corresponding SNOMED code numbers and relevant case numbers.
The karyotypes of a Welsh pony and a Standardbred were 64,XX/64,XY and 63,XO/64,XY respectively. Both intersexes were true hermaphrodites with bilateral ovotestes. Neither intersex showed stallion-like behaviour. Each one had an underdeveloped penis, bilateral seminal vesicles and uterine tissue. It would appear that the chimerism in these equine intersexes resulted from double fertilization or fusion of blastocysts. Mosaicism in the Standardbred is a possibility, resulting from loss of a Y chromosome by anaphase lag in an early embryonic XY cell.
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The single-stranded DNA-binding protein of Escherichia coli significantly alters the strand assimilation reaction catalyzed by recA protein [McEntee, K., Weinstock, G. M. & Lehman, I. R. (1979) Proc. Natl. Acad. Sci. USA 76, 2615--2619]. The binding protein (i) increases the rate and extent of strand assimilation into homologous duplex DNA, (ii) enhances the formation of a complex between recA protein and duplex DNA in the presence of homologous or heterologous single-stranded DNA, (iii) reduces the rate and extent of ATP hydrolysis catalyzed by recA protein in the presence of single-stranded DNA, (iv) reduces the high concentration of recA protein required for strand assimilation, and (v) permits detection of strand assimilation in the presence of the ATP analog, adenosine 5'-O-(O-thiotriphosphate). Single-stranded DNA-binding protein purified from a binding protein mutant (lexC) is considerably less effective than wild-type binding protein in stimulating strand assimilation, a result which suggests that single-stranded DNA-binding protein participates in general recombination in vivo.
A Polled Hereford bull with good libido displayed gross testicular hypoplasia and azoospermia. His left testis weighed 23.6 g, approximately 10% of the testis weight of normal Herefords. Histopathologic examination of the left testis revealed small seminiferous tubules in advanced degeneration and very few with Sertoli cells. The number of Leydig cells was disproportionately large in relation to the seminiferous tubules. From both leucocyte and tissue cultures, the karyotypes were 61, XXY. This bull represents the bovine counterpart of human Klinefelter's syndrome.