A set of BAC clones spanning the human genome

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human fingerprint map, 99% of the current assembled sequence and has an effective resolving power of 79 kb. We have made the clone set publicly available, anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease.     

Evaluation of a selective enrichment technique for the isolation of Campylobacter pylori

To cultivate Campylobacter pylori from contaminated biopsy specimens, Brucella broth was supplemented with 10% fetal calf serum, 1% Vitox, 1000 units/ml polymyxin B sulfate, 10 micrograms/ml vancomycin, and 2 micrograms/ml amphotericin B. Pseudomonas aeruginosa, Candida albicans, and Enterococcus fecalis were cocultivated with C. pylori. All four strains of C. pylori were recoverable at 24 h. When 21 C. pylori strains were studied in pure culture, 86% grew in the selective enrichment medium. In a clinical study, the selective enrichment technique resulted in isolation of C. pylori from 50% of patient samples, compared with isolation from only 36% of samples with agar cultivation. The selective enrichment technique may be more sensitive than techniques currently employed to isolate C. pylori from gastric tissue.     

Evaluation of filters for recovery of Campylobacter jejuni from water.

Campylobacter jejuni has been incriminated in several large waterborne outbreaks, but it has rarely been isolated from water itself. Better methodology is needed for the isolation of C. jejuni from water. We evaluated three types of 0.45-micron microporous filters and three different pore sizes of positively charged depth filters for their ability to recover C. jejuni from seeded, sterile tap and surface water. The microporous filters tested were Millipore HA, Gelman GN6, and Zetapor. Three pore sizes of Zeta Plus depth filters (05S, 30S, and 50S) were evaluated by using an adsorption-elution technique. The overall percent recovery in both tap and surface water by microporous filters was: Zetapor, 66%; Millipore HA, 33%; and Gelman GN6, 33%. Adsorption-elution with Zeta Plus 50S allowed 89% recovery of C. jejuni. These data suggest that both the positively charged Zetapor microporous filter and the Zeta Plus 50S depth filter are effective filters for the recovery of C. jejuni from water.     

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.     

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: II. Distribution of radioactive peptide hormones and hormone precursors in subcellular fractions after pulse and pulse- chase incubation of islet tissue

Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.     

Enterobacteriaceae isolated from iguanid lizards of west-central Texas.

The prevalence of members of the family Enterobacteriaceae in the intestines of seven species of iguanid lizards native to west-central Texas was determined. Of the 67 lizard specimens examined, 48.7% were infected with Salmonella and 9% were infected with Salmonella arizonae. Two lizard species (Sceloporus olivaceus and Crotaphytus collaris) were shown to have a 100% prevalence of Salmonella.     

ELECTRON MICROSCOPIC AND HISTOCHEMICAL COMPARISON OF THE TWO TYPES OF ELECTROPLAQUES OF NARCINE BRASILIENSIS

The torpedine electric fish Narcine brasiliensis has two morphologically distinct electric organs (main and accessory) which also differ with respect to a number of electrophysiological properties. The fine structure of the electroplaques of these organs has been examined by electron microscopy and by a histochemical method for localizing esterase activity with a high degree of resolution. In both kinds of electroplaques the innervated surface (ventral in those of the main organ, dorsal in those of the accessory) is the only site of esterase activity. The latter is further confined to the regions of synaptic contact between vesicle-containing axon terminals and the electroplaque membrane. The synaptic apparatus is similar to, but less elaborate than, that of neuromuscular junctions. The axon terminals and electroplaque membranes are free of connective tissue envelopments. The membrane of the uninnervated surfaces forms a continuum with a dense canalicular network which penetrates deeply into the 7 µ thick electroplaques of the main organ. The canalicular network has about the same thickness in the 20 µ electroplaques of the accessory organ. Except for this difference, the two kinds of cells appear to have the same fine structure. This finding is discussed in relation to the electrophysiological data on functional differences.     

Comparative nuclear and mitochondrial genome diversity in humans and chimpanzees

Restriction mapping and sequencing have shown that humans have substantially lower levels of mitochondrial genome diversity (d) than chimpanzees. In contrast, humans have substantially higher levels of heterozygosity (H) at protein-coding loci, suggesting a higher level of diversity in the nuclear genome. To investigate the discrepancy further, we sequenced a segment of the mitochondrial genome control region (CR) from 49 chimpanzees. The majority of these were from the Pan troglodytes versus subspecies, which was underrepresented in previous studies. We also estimated the average heterozygosity at 60 short tandem repeat (STR) loci in both species. For a total sample of 115 chimpanzees, d = 0.075 +/0 0.037, compared to 0.020 +/- 0.011 for a sample of 1,554 humans. The heterozygosity of human STR loci is significantly higher than that of chimpanzees. Thus, the higher level of nuclear genome diversity relative to mitochondrial genome diversity in humans is not restricted to protein-coding loci. It seems that humans, not chimpanzees, have an unusual d/H ratio, since the ratio in chimpanzees is similar to that in other catarrhines. This discrepancy in the relative levels of nuclear and mitochondrial genome diversity in the two species cannot be explained by differences in mutation rate. However, it may result from a combination of factors such as a difference in the extent of sex ratio disparity, the greater effect of population subdivision on mitochondrial than on nuclear genome diversity, a difference in the relative levels of male and female migration among subpopulations, diversifying selection acting to increase variation in the nuclear genome, and/or directional selection acting to reduce variation in the mitochondrial genome.      

电针介导的基因转移

基因转移是实现基因治疗的关键技术之一 ,目前尚缺少简便、易行、有效、安全的方法 .首次将我国传统的针刺技术与现代转基因技术结合起来 ,创建了一种电针转基因的方法 .应用针灸针携带外源基因 ,经皮针刺 ,进行直流电刺激 ,可实现有效的基因转移 .