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201.
Expression of the human retinoblastoma gene product pp110RB in insect cells using the baculovirus system 总被引:4,自引:0,他引:4
The product of the retinoblastoma susceptibility gene (RB) was overproduced in cultured insect cells using the baculovirus expression system. Upon insertion of the cloned human RB complementary DNA sequence into the viral genome downstream of the promoter of the polyhedrin gene, full-length RB protein with an apparent molecular weight of 110,000 was expressed in the insect cells. This protein was found to be phosphorylated, located in the nuclei of the infected cells, and immunologically indistinguishable from pp110RB of human cells as assayed by several anti-RB antibodies. Following cell disruption and a one-step immunoaffinity chromatographic purification, 6-12 mg of soluble pp110RB with approximately 95% purity were obtained per liter of infected suspension culture. Characterization of the two known biochemical properties of RB protein showed that this purified protein from insect cells behaved similarly to the authentic human pp110RB. First, it bound to DNA, and second, it could form a specific complex with SV40 T antigen in vitro. Prompt translocation of the protein from cytoplasm to nucleus after microinjection further indicated that the purified RB protein may be active. The availability of soluble, intact, and presumably active pp110RB in large quantity represents a significant advance for studying the biochemical and biophysical properties of the RB gene product as well as its potential biological function in cancer suppression. 相似文献
202.
Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers. 总被引:14,自引:3,他引:11 下载免费PDF全文
Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression. 相似文献
203.
Anna L. Trifillis Myong Won Kahng 《In vitro cellular & developmental biology. Plant》1990,26(5):441-446
Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar
to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting
duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase.
Cells were plated on fibronectin-coated culture flasks at a density of 104 live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand
600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure
including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by
the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a
marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic
enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron
origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to
probe pathogenetic mechanisms of potential nephrotoxins.
Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins
Medical Institutions, Baltimore, MD 21205.
This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health
and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate. 相似文献
204.
Growth inhibition of selected food-borne bacteria, particularly Listeria monocytogenes, by plant extracts 总被引:2,自引:1,他引:1
King-Thom Chung W.R. Thomasson Christine D. Wu-Yuan 《Journal of applied microbiology》1990,69(4):498-503
C hung , K.-T., T homasson , W.R. & W u -Y uan , C.D. 1990. Growth inhibition of selected food-borne bacteria, particularly Listeria monocytogenes , by plant extracts. Journal of Applied Bacteriology 69 , 498–503.
Six extracts from Chinese medicinal plants: Tin Men Chu, Sey Lau Pai, Siu Mao Heung, Bak Tao Yung, Kam Chin Chiu and Liao Ya, were tested for their inhibitory effect on selected food-borne bacteria by the well assay technique. Among them, Tin Men Chu, Siu Mao Heung and Sey Lau Pai inhibited the growth of Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, Shigella flexneri, Streptococcus faecalis, Salmonella paratyphi, Salm. enteritidis, Enterobacter aero-genes, Pseudomonas fluorescens, Proteus vulgaris, Alcaligenes faecalis , and three strains of Listeria monocytogenes . Two of these three extracts, Tin Men Chu and Siu Mao Heung, suppressed the growth of L. monocytogenes Scott A in cabbage juice. This inhibition was prevented by the addition of protein but not sodium chloride. Plant extracts show potential to control the growth of food-borne bacteria. 相似文献
Six extracts from Chinese medicinal plants: Tin Men Chu, Sey Lau Pai, Siu Mao Heung, Bak Tao Yung, Kam Chin Chiu and Liao Ya, were tested for their inhibitory effect on selected food-borne bacteria by the well assay technique. Among them, Tin Men Chu, Siu Mao Heung and Sey Lau Pai inhibited the growth of Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, Shigella flexneri, Streptococcus faecalis, Salmonella paratyphi, Salm. enteritidis, Enterobacter aero-genes, Pseudomonas fluorescens, Proteus vulgaris, Alcaligenes faecalis , and three strains of Listeria monocytogenes . Two of these three extracts, Tin Men Chu and Siu Mao Heung, suppressed the growth of L. monocytogenes Scott A in cabbage juice. This inhibition was prevented by the addition of protein but not sodium chloride. Plant extracts show potential to control the growth of food-borne bacteria. 相似文献
205.
Restriction site mapping requires a generator to put forwardpossible maps and a constraint checker to reject false maps.Ideally these combine to give an algorithm which calculatesa sound and complete solution set. Three algorithms for generationare presented and compared. Two decompose a multi-enzyme problem(3) into subproblems. The constraint checker is based on separationtheory. Some insights into the extent of constraint checkinginvolved in and the feasibility of more checking for three ormore enzymes are discussed. The trade-off between computationtime and the soundness of the solution set is examined.
Received on July 30, 1989; accepted on April 4, 1990 相似文献
206.
Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection. 总被引:1,自引:1,他引:0 下载免费PDF全文
A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied. 相似文献
207.
Inter- and intraclade neutralization of human immunodeficiency virus type 1: genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes. 总被引:22,自引:18,他引:4 下载免费PDF全文
We have studied genetic variation among clades A through E of human immunodeficiency virus type 1 (HIV-1) at the levels of antibody binding to gp120 molecules and virus neutralization. We are unable to identify neutralization serotypes that correspond to the genetic clades. Instead, we observe that inter- and intraclade neutralization of primary isolates by HIV-1-positive sera is generally weak and sporadic; some sera show a reasonable degree of neutralization breadth and potency whereas others are relatively sensitive to neutralization, but no consistent pattern was found. However, a few sera were able to neutralize across clades with significant potency, an observation which may have implications for the feasibility of a broadly effective HIV-1 vaccine involving humoral immunity. Serological assays measuring anti-gp120 antibody binding also failed to identify serotypes that correspond precisely to the genetic clades, but some indications of clade-specific binding were observed, notably with sera from clades B and E. A representative protein for each clade (A through E) was selected on the basis of its specificity, defined as high seroreactivity with sera from individuals infected with virus of that clade and lower reactivity with sera from individuals infected with viruses from other clades. The seroreactivity patterns against these five proteins could be used to predict the genotype of the infecting virus with moderate success. 相似文献
208.
Hae-Jin Chung Christopher Shaffer Ross MacIntyre 《Molecular & general genetics : MGG》1996,250(5):635-646
InDrosophila, unlike humans, the lysosomal acid phosphatase (Acph-1) is a non-essential enzyme. It is also one of the most rapidly evolving gene-enzyme systems in the genus. In order to determine which parts of the enzyme are conserved and which parts are apparently under little functional constraint, we cloned the gene fromDrosophila melanogaster via a chromosomal walk. Fragments from the gene were used to recover an apparently full-length cDNA. The cDNA was subcloned into aDrosophila transformation vector where it was under the control of the 5 promoter sequence of thehsp-70 gene. Three independent transformants were obtained; in each, Acph-1 expression from the cDNA was constitutive and not dependent on heat shock, as determined by densitometric analyses of the allozymic forms of the enzyme. The pattern of expression indicates thehsp-70 and endogenousAcph-1 promoters act together in some, but not all, tissues. The sequence of the cDNA was determined using deletions made with exonuclease III, and primers deduced from the cDNA sequence were used to sequence the genomic clone. Five introns were found, and putative 5 up-stream regulatory sequences were identified. Amino acid sequence comparisons have revealed several highly conserved motifs betweenDrosophila Acph-1 and vertebrate lysosomal and prostatic acid phosphatases. 相似文献
209.
Dong-Uk Kim Seung-Kiel Park Kyung-Sook Chung Myung-Un Choi Hyang-Sook Yoo 《Molecular & general genetics : MGG》1996,252(1-2):20-32
ASchizosaccharomyces pombe homolog of mammalian genes encoding G protein subunits,gpb1
+, was cloned by the polymerase chain reaction using primer pairs that correspond to sequences conserved in several G genes of other species followed by screening of genomic and cDNA libraries. Thegpb1 gene encodes 317 amino acids that show 47% homology with human G
1 and G
2 and 40% homology withSaccharomyces cerevisiae G protein. Disruption of thegpb1 gene indicated that this gene is not required for vegetative cell growth. However,gpb1-disrupted haploid cells mated and sporulated faster than wild-type cells, both in sporulation (MEA) and in complex medium (YE): when examined 23 h after transfer to sporulation medium, 35% ofgpb1-disrupted haploid pairs had undergone conjugation and sporulation, whereas only 3–5% of wild-type haploid pairs had done so. Overexpression of thegpb1 gene suppressed this facilitated conjugation and sporulation phenotype ofgpb1-disrupted cells but did not cause any obvious effect in wild-type cells. Co-disruption of one of the twoS. pombe G-subunit genes,gpa2, in thegpb1-disrupted cells did not change the accelerated conjugation and sporulation phenotype of thegpb1
– cells. However, co-disruption of theras1 gene abolished thegpb1
– phenotype. These results suggest that Gpbl is a negative regulator of conjugation and sporulation that apparently works upstream of Ras1 function inS. pombe. The possible relationship of Gpbl to two previously identified, putative G proteins ofS. pombe is discussed.A preliminary report of this work first appeared in an abstract of the Genetic Society of America, 1993 Yeast Genetics and Molecular Biology Meeting, p. 92 and was presented at the American Association of Cancer special meeting on Cell Signalling and Cancer Treatment, 1993 相似文献
210.
The frequencies of chromosome aberrations in 135 workers from nuclear-power plants were compared with those in 135 age-matched controls. A total of 135,000 cells was scored. The frequencies of dicentric chromosome were 1.67 × 10−3 in the exposed group and 0.49 × 10−3 in the control group and those of chromosome-type deletion were 3.33 × 10−3 and 1.10 × 10−3, respectively. The frequencies of all types of chromosome aberrations in the exposed subjects were higher than those in the control group, but no significant trend of dose-dependent increase was observed when only the exposed group were considered. Poisson regression analysis, with both exposed and control included, showed that there was a significant association of chromosome aberration with radiation dose and the duration of work, but not with age, smoking habit and alcohol intake. It was also found that recent exposure to radiation, within the last 5 years, had contributed more to the observed chromosome aberration than earlier exposure. 相似文献