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21.
Peter Hechtman Bernard Boulay Marc De Braekeleer Eve Andermann Serge Melançon Jean Larochelle Claude Prevost Feige Kaplan 《Human genetics》1992,90(4):402-406
Mutations at the hexosaminidase A (HEXA) gene which cause Tay-Sachs disease (TSD) have elevated frequency in the Ashkenazi Jewish and French-Canadian populations. We report a novel TSD allele in the French-Canadian population associated with the infantile form of the disease. The mutation, a GA transition at the +1 position of intron 7, abolishes the donor splice site. Cultured human fibroblasts from a compound heterozygote for this transition (and for a deletion mutation) produce no detectable HEXA mRNA. The intron 7+1 mutation occurs in the base adjacent to the site of the adult-onset TSD mutation (G805A). In both mutations a restriction site for the endonuclease EcoRII is abolished. Unambiguous diagnosis, therefore, requires allele-specific oligonucleotide hybridization to distinguish between these two mutant alleles. The intron 7+1 mutation has been detected in three unrelated families. Obligate heterozygotes for the intron 7+1 mutation were born in the Saguenay-Lac-St-Jean region of Quebec. The most recent ancestors common to obligate carriers of this mutation were from the Charlevoix region of the province of Quebec. This mutation thus has a different geographic centre of diffusion and is probably less common than the exon 1 deletion TSD mutation in French Canadians. Neither mutation has been detected in France, the ancestral homeland of French Canada. 相似文献
22.
J L Hopkins R Betageri K A Cohen M J Emmanuel C R Joseph P M Bax P V Pallai M T Skoog 《Journal of biochemical and biophysical methods》1991,23(2):107-113
The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis. 相似文献
23.
Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites. 总被引:3,自引:2,他引:1
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J T Millard M F Weidner J J Kirchner S Ribeiro P B Hopkins 《Nucleic acids research》1991,19(8):1885-1891
A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency. 相似文献
24.
Two membrane filter (MF) and two most-probable-number methods for enumerating Vibrio parahaemolyticus were compared. The MF methods used elevated-temperature incubations (41 and 42 degrees C) and were more specific than the most-probable-number methods (conducted at 35 degrees C). The MF method with a hydrophobic grid and a repair step was most effective. 相似文献
25.
Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters. 总被引:4,自引:3,他引:1
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Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters. 相似文献
26.
Role of triglyceride-rich lipoproteins and hepatic lipase in determining the particle size and composition of high density lipoproteins 总被引:1,自引:0,他引:1
These studies examined the proposition that the small particle size of HDL3 in the plasma of hypertriglyceridemic subjects is the consequence of the sequential actions of lipid transfer protein and hepatic lipase on HDL. Incubation of unmodified total HDL or HDL3 in the presence of hepatic lipase resulted in a depletion of phospholipid, but little change in the size of the particles. On the other hand, HDL3 that had first been depleted of cholesteryl ester and enriched with triglyceride and phospholipid, during prior incubation with Intralipid and a source of lipid transfer protein, were much more susceptible to the action of hepatic lipase. When these modified HDL3 were incubated with hepatic lipase there was a depletion of the triglyceride and phospholipid content and a conversion into much smaller particles the same size as those predominant in hypertriglyceridemic subjects. These very small particles were derived from a population of modified particles that were larger than the original HDL3 and were within the size range of HDL2. It is proposed, therefore, that in the plasma of hypertriglyceridemic subjects there exists a dynamic balance between the formation of enlarged triglyceride-rich HDL and a secondary conversion of these particles by hepatic lipase to form populations of very small HDL. 相似文献
27.
Genetic and physical characterization of IncM plasmid pBWH1 and its variance among natural isolates 总被引:2,自引:0,他引:2
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J D Hopkins K H Mayer E S Gilleece T F O''Brien M Syvanen 《Journal of bacteriology》1986,165(1):47-52
We present a genetic and physical characterization of the IncM plasmid pBWH1. A physical map was constructed for the enzymes EcoRI, BamHI, SalI, BglII, HindIII, MstII, and XhoI. A series of deletions and a series of subclones of pBWH1 were constructed and used to determine the locations on this map of the transfer region; the replication region; and the genes determining resistance to beta-lactams, chloramphenicol, the sulfonamides, and gentamicin. We compared 51 different isolates, including isolates which had lost individual antibiotic resistances or the transfer phenotype, and showed that variations occurred in all regions of the plasmid genome. Frequently, correlations could be made between phenotypic variation and variation of the EcoRI fragments which contained the gene determining that phenotype. 相似文献
28.
Properties of a serine hydroxymethyltransferase in which an active site histidine has been changed to an asparagine by site-directed mutagenesis 总被引:1,自引:0,他引:1
Histidine 228 at the active site of Escherichia coli serine hydroxymethyltransferase was replaced with an asparagine. The mutant enzyme was expressed in a strain of E. coli that lacks wild type enzyme. Absorption spectra, circular dichroism spectra, and differential scanning calorimetry thermograms suggest that the amino acid change at the active site causes no detectable change in the tertiary structure of the enzyme. Kinetic studies demonstrated that kcat for the mutant enzyme is about 25% of the value for the wild type enzyme with either L-serine or allothreonine as substrate. Km or Kd values for amino acid substrates and reduced folate compounds were 2-10-fold larger with the mutant enzyme. The rate of interconversion of several enzyme-glycine complexes showed that the conversion of the external aldimine to the quinoid complex is not the rate-determining step for either the mutant or wild type enzyme in the presence of tetrahydrofolate. The binding of L-serine to the wild type enzyme gives a more thermally stable enzyme and increases its affinity for tetrahydrofolate. These effects are not found when L-serine binds to the mutant enzyme. The studies demonstrate that histidine 228 is not a catalytically essential residue and suggest that it is involved in interacting with either the amino acid substrate or the enzyme-bound pyridoxal phosphate. 相似文献
29.
Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells 总被引:13,自引:7,他引:6
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K Miller J Beardmore H Kanety J Schlessinger C R Hopkins 《The Journal of cell biology》1986,102(2):500-509
We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4 degrees C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37 degrees C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20 degrees C the ligand-receptor complex accumulated in the pericentriolar compartment. Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step. 相似文献
30.