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Biomedical subjects

L Jin

Publications and source records attributed to L Jin.

At least 307 records · Page 17Linked to original sources
PubMed1997

In situ hybridization: methods and applications.

In situ hybridization (ISH) combines molecular biological techniques with histological and cytological analysis of gene expression. RNA and DNA can be readily localized in specific cells with this method. ISH has been useful as a research tool, and recent studies have used this technique in the diagnostic pathology laboratory and in microbiology for the tissue localization in infectious agents. Other recent developments in the applications of ISH involve in situ polymerase chain reaction (PCR) and in situ reverse transcription (RT)-PCR, which can be used to detect very low levels of nucleic acids in tissues by taking advantage of the powerful amplification capacity of PCR. In situ PCR will contribute significantly to progress in this field because of the marked increase in the sensitivity of this method.

Cytogenetics↗
PubMed1997

Aberrant p27kip1 expression in endocrine and other tumors.

The p27kip1 (p27) gene encodes an inhibitor of cyclin-dependent kinase activity. The expression of p27 protein in normal and neoplastic tissues was investigated by immunoblotting and immunohistochemistry. Immunoblotting studies detected a 27-kd protein band that was decreased in neoplastic pituitary tissues compared with normal pituitary. Immunostaining of 177 tissues showed abundant expression of p27 protein in normal tissues with decreased numbers of immunoreactive cells in adenomas and carcinomas in both endocrine and nonendocrine tissues. p27 expression was inversely related to the proliferation marker Ki-67 antigen detected with monoclonal antibody MIB-1. Parathyroid adenomas and hyperplasias had similar Ki-67 labeling indices; however, hyperplasias had threefold more p27-positive cells than parathyroid adenomas, suggesting that p27 immunostaining may be useful in distinguishing between these two conditions. These results indicate that there is widespread aberrant p27 expression in hyperplastic tissues and in benign and malignant neoplasms compared with normal tissues. Immunohistochemical analysis of p27 along with Ki-67 may be used to assess the biological behavior of various neoplasms, to classify hyperplastic and neoplastic tissues, and to study cell cycle regulation during tumor progression.

Cell Cycle Proteins↗
PubMed1997

Ethnic-affiliation estimation by use of population-specific DNA markers.

During the past 10 years, DNA analysis has revolutionized the determination of identity in a forensic context. Statements about the biological identity of two human DNA samples now can be made with complete confidence. Although DNA markers are very powerful for distinguishing among individuals, most offer little power to distinguish ethnicity or to support any statement about the physical characteristics of an individual. Through a search of the literature and of unpublished data on allele frequencies we have identified a panel of population-specific genetic markers that enable robust ethnic-affiliation estimation for major U.S. resident populations. In this report, we identify these loci and present their levels of allele-frequency differential between ethnically defined samples, and we demonstrate, using log-likelihood analysis, that this panel of markers provides significant statistical power for ethnic-affiliation estimation. In addition to their use in forensic ethnic-affiliation estimation, population-specific genetic markers are very useful in both population- and individual-level admixture estimation and in mapping genes by use of the linkage disequilibrium created when populations hybridize.

Alleles↗
PubMed1997

Scatter factor stimulates tumor growth and tumor angiogenesis in human breast cancers in the mammary fat pads of nude mice.

Scatter factor (SF) (also known as hepatocyte growth factor) is a plasminogen-related growth factor that induces tumor cell motility, invasion, and angiogenesis. Its receptor is a tyrosine kinase encoded by c-met, a protooncogene. Human breast cancer cells express SF and c-met in vivo; but human breast cancer cell lines do not produce SF in vitro. To determine whether SF can modulate the in vivo growth of human breast cancers within a natural mammary environment, we studied the orthotopic growth of SF-transfected (SF+) versus control (SF-) clones of MDAMB231 human mammary carcinoma cells in the mammary fat pads of athymic nude mice. SF+ clones expressed SF mRNA and produced very high titers of SF protein, whereas SF- clones did not express SF mRNA or produce detectable SF protein. Two SF+ clones (21 and 29) showed significantly increased tumor growth rates, reaching 3- to 4-fold larger primary tumor volumes and weights by time of killing (p < 0.001), as well as higher rates of axillary lymph node metastasis (p < 0.02), as compared with two SF- clones (32 and 34). In contrast, in vitro proliferation rates, two-dimensional colony formation, and soft agar colony formation were no greater in SF+ than in SF- clones. We performed further studies to investigate the discrepancy between the in vivo and in vitro growth results. Tumor extracts from SF+ clone (21 + 29) tumors had 50-fold higher SF content than did SF- clone (32 + 34) tumors, confirming high-level SF expression in vivo in SF+ tumors. Immunostaining of tumor sections for proliferating cell nuclear antigen revealed only a modest increase in the proportion of cycling cells in SF+ versus SF- tumors (70% versus 60%, respectively). The terminal deoxytransferase-labeling index was equally low (approximately 1%) in SF+ and SF- tumors, suggesting that apoptosis was not responsible for the slower growth of SF- tumors. However, SF+ tumors had significantly higher tumor microvessel densities than SF- tumors (p < 0.001). Moreover, there were much higher titers of chemotactic activity for microvascular endothelial cells in cell-conditioned media and primary tumor extracts from SF+ clones as compared with SF- clones. As demonstrated using the rat cornea assay, there was more angiogenic activity in SF+ tumor extracts than in SF- extracts. The increased chemotactic and angiogenic activities in SF+ tumor extracts were not explained by secondary alterations in the content of the angiogenic mediator, vascular endothelial growth factor, or the antiangiogenic glycoprotein, thrombospondin-1; and those activities were neutralized using an anti-SF monoclonal antibody. These findings suggest that SF promotes the orthotopic growth of human breast cancers, at least in part, by stimulating tumor angiogenesis.

Adipose Tissue↗
PubMed1997

Defining microsatellite alleles by genotyping global indigenous human populations and non-human primates.

Polymorphisms at variable number of tandem repeat (VNTR) loci have been used in forensic science for almost decade. Microsatellite loci, especially tri-, tetra-, and pentanucleotide repeat loci, have shown great potential in personal identification and paternity testing. In this report, we describe results of genotyping three tetranucleotide repeat loci (D5S818, D7S820, D13S317) in 16 worldwide indigenous human populations and one chimpanzee population which were being developed for forensic applications. We demonstrate the utility of typing globally diverse populations in defining microsatellite alleles: Specifically (i) investigating the measurement errors of each allele using semi-automatic genotyping instrumentation and software, (ii) assessing the range of alleles, (iii) understanding the extent of allele frequency differences across worldwide populations, and (iv) identifying possible anomalous alleles with complex structures.

Alleles↗
PubMed1997

Studies on the metabolic activation of disulfiram in rat. Evidence for electrophilic S-oxygenated metabolites as inhibitors of aldehyde dehydrogenase and precursors of urinary N-acetylcysteine conjugates.

Recent studies on the mechanism by which disulfiram inhibits aldehyde dehydrogenase have provided evidence for the formation of reactive intermediates that are thought to carbamoylate, and thereby inactivate the enzyme. In our study, rats were dosed with either disulfiram (0.25 mmol kg-1 i.p.) or its reduced metabolite diethyldithiocarbamate (DDTC; 0.5 mmol kg-1 i.p.) and urine was collected for the analysis of metabolites derived from putative reactive intermediates. By means of ionspray LC-MS/MS, two novel N-acetylcysteine (NAC) conjugates, i.e., N-acetyl-S-(N, N-diethylcarbamoyl)cysteine and N-acetyl-S-(N, N-diethylthiocarbamoyl)cysteine, were identified in urine specimens. Quantitative analyses indicated that, over the 0- to 24-hr period after drug administration, urinary excretion of N-acetyl-S-(N, N-diethylcarbamoyl)cysteine accounted for 7.5 +/- 4.0 and 6.2 +/- 1.0%, respectively, of the dose of disulfiram and diethyldithiocarbamate, while the corresponding thiocarbamoyl conjugate, N-acetyl-S-(N, N-diethylthiocarbamoyl)cysteine, accounted for a further 0.5 +/- 0.3 and 0.3 +/- 0.1%, respectively, of the dose. These conjugates are believed to derive from reactive sulfoxide and sulfone metabolites of disulfiram, namely S-methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO), S-methyl-N, N-diethylthiocarbamate sulfone (DETC-MeSO2), S-methyl-N, N-diethyldithiocarbamate sulfoxide (DDTC-MeSO) and S-methyl-N, N-diethyldithiocarbamate sulfone (DDTC-MeSO2), which were found to carbamoylate N-acetylcysteine in vitro with the following rank order of reactivity: DDTC-MeSO2 > DETC-MeSO2 > DDTC-MeSO > DETC-MeSO. In vitro experiments with aldehyde dehydrogenase showed that all four S-oxygenated metabolites inhibited the enzyme effectively. Furthermore, inclusion of NAC in incubation media attenuated significantly the inhibition by DDTC-MeSO2, DETC-MeSO2 and DDTC-MeSO, but had little effect on that by DETC-MeSO. Our results are consistent with the hypothesis that disulfiram and diethyldithiocarbamate undergo activation by a sequence of metabolic reactions leading to the formation of electrophilic S-methyl sulfoxides and sulfones that carbamoylate, and thereby inhibit, aldehyde dehydrogenase and possibly other enzymes.

Acetylcysteine↗
PubMed1997

Biochemical and biophysical characterization of human recombinant lecithin: cholesterol acyltransferase.

We established a Chinese hamster ovary cell line that constitutively expresses up to 5 mg/L of human recombinant lecithin: cholesterol acyltransferase (rLCAT). We purified the rLCAT to > 96% purity, and characterized it along with plasma LCAT (pLCAT) biochemically and biophysically. The recombinant enzyme is more heavily glycosylated and more heterogeneous in its carbohydrate content than the plasma enzyme, as revealed by differences in molecular weight and pI isoforms, determined by mass spectrometry and isoelectric focusing. Recombinant LCAT is half as active enzymatically as pLCAT. The difference in activity is due to differences in the catalytic rates rather than in the apparent K(m) values, suggesting that the binding of the rLCAT to interfaces is not altered by its different glycosylation pattern. Despite these differences, rLCAT has essentially the same intrinsic tryptophan fluorescence emission spectrum and far-UV CD spectrum as pLCAT, indicating that the tertiary and secondary structures of both enzyme forms are very similar. Both enzyme forms have a propensity to self-associate, and their multimers appear resistant to dissociation by SDS and dilution. The free energies of unfolding (delta G(H2O)) of rLCAT and pLCAT are 3.4 +/- 0.2 and 3.2 +/- 0.2 kcal/mol, respectively, as determined by guanidine hydrochloride denaturation monitored by fluorescence. These relatively low delta G(H2O) values support the notion that LCAT is capable of undergoing major conformational changes upon interaction with interfacial substrates.

Animals↗
PubMed1997

Transforming growth factor-beta, transforming growth factor-beta receptor II, and p27Kip1 expression in nontumorous and neoplastic human pituitaries.

Transforming growth factor (TGF)-beta has been implicated in the regulation of normal and neoplastic anterior pituitary cell function. TGF-beta regulates the expression of various proteins, including p27Kip1 (p27), a cell cycle inhibitory protein. We examined TGF-beta, TGF-beta type II receptor (TGF-beta-RII), and p27 expression in normal pituitaries, pituitary adenomas, and carcinomas to analyze the possible roles of these proteins in pituitary tumorigenesis. Normal pituitary, pituitary adenomas, and pituitary carcinomas all expressed TGF-beta and TGF-beta-RII immunoreactivity. Reverse transcription polymerase chain reaction analysis showed TGF-beta 1, -beta 2, and -beta 3 isoforms and TGF-beta-RII in normal pituitaries and pituitary adenomas. Pituitary adenomas cells cultured for 7 days in defined media showed a biphasic response to TGF-beta with significant inhibition of follicle-stimulating hormone secretion at higher concentrations (10(-9) mol/L) and stimulation of follicle-stimulating hormone secretion at lower concentrations (10(-13) mol/L) of TGF-beta 1 in gonadotroph adenomas. Immunohistochemical analysis for p27 protein expression showed the highest levels in nontumorous pituitaries with decreased immunoreactivity in adenomas and carcinomas. When nontumorous pituitaries and various adenomas were analyzed for p27 and specific hormone production, growth hormone, luteinizing hormone, and thyroid-stimulating hormone cells and tumors had the highest percentages of cells expressing p27, whereas adrenocorticotrophic hormone cells and tumors had the lowest percentages. Immunoblotting analysis showed that adrenocorticotrophic hormone adenomas also had the lowest levels of p27 protein. Semiquantitative reverse transcription polymerase chain reaction and Northern hybridization analysis did not show significant differences in p27 mRNA expression in the various types of adenomas or in nontumorous pituitaries. In situ hybridization for p27 mRNA showed similar distributions of the gene product in nontumorous pituitaries, pituitary adenomas, and carcinomas. These results indicate that TGF-beta and TGF-beta-RII are widely expressed in nontumorous pituitaries and in pituitary neoplasms and that TGF-beta 1 regulates pituitary hormone secretion. The levels of the TGF-beta-regulated protein p27 decreases in the progression of normal to neoplastic pituitaries. In contrast, the mRNA levels of p27 remained relatively constant in nontumorous pituitaries, pituitary adenomas, and carcinomas, indicating that p27 protein levels in adenomas and carcinomas are regulated by translational and post-translational mechanisms.

Adenoma↗
PubMed1997

Kallikrein gene therapy in newborn and adult hypertensive rats.

The tissue kallikrein-kinin system has been postulated to play an important role in blood pressure regulation. Kallikreins are serine proteinases that release potent vasodilating kinin peptides from precursor kininogens by limited proteolysis. Our recent studies show that systemic delivery of the human tissue kallikrein gene into adult spontaneously hypertensive rats (SHR) results in a sustained reduction of blood pressure for several weeks. The goal of this study is to evaluate whether early delivery of the kallikrein gene into newborn SHR could exert a suppressive effect on blood pressure phenotype during rat growth and development. A human tissue kallikrein cDNA construct, under the control of cytomegalovirus promoter (CMV-cHK), or vector DNA was injected subcutaneously into the necks of 2-day-old SHR. Blood pressures were monitored biweekly from 3 to 19 weeks by the tail-cuff method. A single injection of the human kallikrein cDNA construct caused a significant reduction of blood pressure (n = 6, p < 0.001) from 11 to 17 weeks after injection compared with control rats receiving vector DNA. Intravenous delivery of the human tissue kallikrein gene into adult SHR produced blood pressure lowering effects (n = 6, p < 0.001) that lasted for 6 weeks in male but not in female rats. The expression of human tissue kallikrein in rats was identified by reverse transcription polymerase chain reaction followed by Southern blot analysis and an ELISA specific for human tissue kallikrein. Kallikrein gene delivery did not cause any changes in body weight, urine volume, or water intake in the experimental animals compared with the control group. No antibodies to either human tissue kallikrein or its DNA were detected in rat sera 19 weeks postinjection. These results show that delivery of the kallikrein gene at an early stage of life has a protective effect against development of hypertension in adult SHR and that gender differences could be a factor in kallikrein gene therapy for the treatment of hypertensive disorders.

Aging↗
PubMed1997

Hepatitis G virus infection in hemodialysis patients and the effects of interferon treatment.

OBJECTIVE: To characterize the nature of hepatitis G virus (HGV) infections in hemodialysis patients and to determine the responsiveness of HGV to antiviral therapy in these patients. METHODS: HGV, a recently identified flavivirus, is associated with non-A-E viral hepatitis infections. We studied HGV infections in hepatitis C virus (HCV)-infected hemodialysis patients over a 1-yr period, using two independent PCR assays and nucleic acid sequencing. Thirty-four of 63 study patients were treated with interferon. RESULTS: We observed a 27% prevalence (17/63 patients) and a 4% annual incidence of HGV infections in the study population. HGV was not detected in any of the 10 HGV-infected patients immediately after interferon therapy. Although seven of these 10 patients developed HGV relapses, three had long-term responses. The interferon responsiveness of HGV and HCV appeared to be unrelated. In contrast, all seven untreated HGV-infected patients remained viremic. Sequence analyses of the different HGV isolates revealed only very limited genetic variability in the polymerase chain reaction-amplified regions of HGV during 1 yr of observation. CONCLUSIONS: Our data suggest that HCV-infected hemodialysis patients are at substantial risk of acquiring HGV infection and that HGV infections are prevalent in this population. In addition, HGV infections become chronic but are responsive to interferon treatment.

Adult↗
PubMed1996

Mutation rate varies among alleles at a microsatellite locus: phylogenetic evidence.

The understanding of the mutational mechanism that generates high levels of variation at microsatellite loci lags far behind the application of these genetic markers. A phylogenetic approach was developed to study the pattern and rate of mutations at a dinucleotide microsatellite locus tightly linked to HLA-DQB1 (DQCAR). A random Japanese population (n = 129) and a collection of multiethnic samples (n = 941) were typed at the DQB1 and DQCAR loci. The phylogeny of DQB1 alleles was then reconstructed and DQCAR alleles were superimposed onto the phylogeny. This approach allowed us to group DQCAR alleles that share a common ancestor. The results indicated that the DQCAR mutation rate varies drastically among alleles within this single microsatellite locus. Some DQCAR alleles never mutated during a long period of evolutionary time. Sequencing of representative DQCAR alleles showed that these alleles lost their ability to mutate because of nucleotide substitutions that shorten the length of uninterrupted CA repeat arrays; in contrast, all mutating alleles had relatively longer perfect CA repeat sequences.

Alleles↗
PubMed1996

Expression of calbindin-D28K by reactive astrocytes in gerbil hippocampus after ischaemia.

Calbindin-D28K (Calbindin) is a member of the superfamily of calcium-binding proteins that is implicated in the regulation of intracellular calcium. In the adult mammalian brain, calbindin was thought to be present only in neurones, where it is believed to serve a neuroprotective role. We now report the expression of calbindin after ischaemia in reactive astrocytes in the CA1 subfield of the hippocampus. Since other calcium-binding proteins, such as S-100 and calmodulin, which induce transformation or proliferation of glia, occur in astrocytes, it is conceivable that the expression of calbindin after ischaemia might be an important part of the process of gliosis.

Animals↗
PubMed1996

Crystal structure of an acidic neurotoxin from scorpion Buthus martensii Karsch at 1.85 A resolution.

The crystal structure of an acidic scorpion neurotoxin, BmK M8, purified from Chinese scorpion Buthus martensii Karsch (BmK), has been determined by the molecular replacement method. It is the first structure of an acidic alpha-scorpion neurotoxin reported so far. The crystals adopt a symmetry of space group P2(1) and contain one molecule per asymmetric unit. The structure has been refined to an R factor of 18.1% using reflection data in the range of 8 to 1.85 A resolution, with standard deviations from ideal geometry of 0.017 A and 2.43 degrees for bond length and angle, respectively. The 12 residues at the C terminus with unknown sequence were determined by crystallographic refinement. The refined model shows that the structural core, consisting of a motif beta alpha beta beta, is similar to that of toxin II from Androctonus australis Hector (AaH II) or Variant 3 from Centruroides sculpturatus Ewing (CsE V3). The three conformationally variable loops protruding from this structural core are different from that of AaH II, and especially from that of CsE V3. Compared with the most potent and basic alpha-toxin AaH II, the BmK M8 is a relatively inactive toxin (1100 times less active than AaH II) with an unusually low isoelectric point (pI 5.3). Sequence alignment of the two toxins shows a difference of 26 residues (40.6%). Among them four basic or neutral residues in AaH II, namely Val10, Lys28, Val55 and Gly59, are changed to acidic glutamate in BmK M8. The residues Glu10, Glu28 and Glu55 of BmK M8 are located on a surface (Face B), opposite the "conserved hydrophobic surface" (Face A). The latter is a functionally important area proposed by Fontecilla-Camps et al. Our observations suggest that in addition to Face A, Face B may also be involved in the biological activity of scorpion toxins. The structure of BmK M8 shows an evident conformational change of the alpha-amino group at the N terminus and a deorganization of Arg2 caused by the mutation D53A. These structural changes may also be responsible for the weak toxicity of BmK M8. In association with the information from chemical modifications, a multisite binding mode for toxin-receptor interaction and three "toxic regions" in relevance to the binding process, including Face A, Face B and Site C, are proposed. Face A, mainly consisting of Tyr5, 35, 47, the alpha-amino group, Arg2 and Asp3, may be more essential for the binding. Face B, mainly comprising conserved residues Tyr14, 21, Lys28 and Val55, may contribute to the high efficacy of the binding process and substitutions by acidic residues in this area could strongly weaken the toxic activity. Site C, formed by Lys58 and Arg62 at the C terminus and Arg41 and Tyr42 from loop 38-44, may be involved in binding site specificity.

Amino Acid Sequence↗
PubMed1996

Scatter factor expression and regulation in human glial tumors.

Scatter factor (SF) (also known as hepatocyte growth factor [HGF]) is a cytokine that induces cell motility in vitro and angiogenesis in vivo. SF appears to be a determinant of the malignant phenotype in certain systemic cancers. We detected SF in extracts prepared from human gliomas, with the highest levels found in malignant tumors. Human glioblastoma cells expressed both SF and its receptor (c-met protein) in vivo, as demonstrated by immunohistochemistry. Consistent with these observations, we found moderate to high levels of production of immunoreactive and biologically active SF by cultured human glioblastoma cells (3 of 8 lines) and by neural microvascular endothelial cells (NMVEC) (3 of 3 lines). SF stimulated the proliferation of glioblastoma and NMVEC cell lines by paracrine or autocrine mechanisms. Conditioned medium (CM) from both glioblastoma and NMVEC cells contained SF-inducing factor (SF-IF) activity, defined by its ability to stimulate SF production in an indicator cell line (MRC5 human fibroblasts). This activity consisted of a high-molecular-weight (> 30 kDa), heat-sensitive component and a low-molecular weight (< 30 kDa), heat-stable component. Furthermore, glioblastoma CM stimulated NMVEC SF production, and NMVEC CM stimulated glioblastoma cell SF production, by 3- to 6-fold in each case. Our findings demonstrate that SF-dependent interactions between glioma cells, and between glioma cells and endothelium, can contribute to the heterogeneous proliferative and angiogenic phenotypes of malignant gliomas in vivo.

Brain↗
PubMed1996

Systemic and portal vein delivery of human kallikrein gene reduces blood pressure in hypertensive rats.

There is an inverse correlation between systemic blood pressure and urinary kallikrein levels in humans and hypertensive animal models, suggesting that the tissue kallikrein-kinin system plays an important role in blood pressure regulation. In this study, we explored the potential of human kallikrein gene delivery on blood pressure reduction in spontaneously hypertensive rats (SHR). The human tissue kallikrein gene or cDNA was placed under the control of following promoters: the metallothionein gene metal response-element (MRE-pHK), albumin gene (ALB-pHK), Rous sarcoma virus 3' long terminal repeat (LTR) (RSV-cHK), and cytomegalovirus (CMV-cHK). A single injection of these kallikrein DNAs results in a significant reduction of blood pressure in SHR, which lasts for 5-6 weeks. Systemic delivery of CMV-cHK, RSV-cHK, and MRE-pHK has a greater effect on blood pressure reduction than ALB-pHK, whereas intraportal vein gene delivery of ALB-pHK is more effective than the other kallikrein DNA constructs. The degree of blood pressure reduction depends on the amount of administered DNA and the age of the animals. Reduction of blood pressure was observed in adult, but not young, SHR. The expression of human tissue kallikrein in rats was identified by an ELISA that is specific for human tissue kallikrein. No antibodies to either human tissue kallikrein or its DNA were detected in rat sera after somatic gene delivery. These results show that somatic gene delivery of human tissue kallikrein causes a lowering effect of systolic blood pressure in genetically hypertensive rats and provide valuable information for kallikrein gene therapy in the treatment of hypertension.

Animals↗
PubMed1996

In vivo and in vitro studies of TrpR-DNA interactions.

The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively using in vitro and in vivo methods. DNA sequence requirements for 1:1 and tandem 2:1 (TrpR:DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one helix-turn-helix motif of one TrpR dimer is 3'CNTGA5'5'GNACT3', consistent with contacts observed by X-ray diffraction analysis of cocrystalline 1:1 and 2:1 complexes. Half-sites can be paired to form a palindrome either by direct abutment, forming the nucleation site for a tandem 2:1 complex, or with an 8-base-pair spacer, forming a 1:1 target. Dimethylsulfate (DMS) methylation-protection footprinting in vitro of 1:1 and 2:1 complexes formed sequentially on the two unequal half-site pairs of the trpEDCBA operator from Serratia marcescens indicated an obligate hierarchy of site occupancy, with one half-site pair serving as the nucleation site for tandem binding. DMS footprinting of Escherichia coli operators in vivo showed that, over a wide range of intracellular TrpR concentration, the trpEDCBA operator is occupied by three repressor dimers, aroH is occupied by two dimers, and the 1:1 binding mode is used on the trpR operator. The coexistence of these distinct occupancy states implies that changes in protein concentration affect only the fractional occupancy of each operator rather than the binding mode, which is determined by the number of half-site sequences present in the operator region. Cooperativity of tandem complex formation measured by gel retardation using a symmetrized synthetic operator containing identical, optimal sites spaced as in natural operators was found to be modest, implying a maximum coupling free energy of approximately -2 kcal/mol. On other sequences the apparent degree of cooperativity, as well as the apparent affinity, varies with sequence and sequence context in a manner consistent with the structural models and which suggests compensation between affinity and cooperativity as a mechanism that allows tolerance of operator sequence variation.

Bacterial Proteins↗
PubMed1996

A pre-Columbian Y chromosome-specific transition and its implications for human evolutionary history.

A polymorphic C-->T transition located on the human Y chromosome was found by the systematic comparative sequencing of Y-specific sequence-tagged sites by denaturing high-performance liquid chromatography. The results of genotyping representative global indigenous populations indicate that the locus is polymorphic exclusively within the Western Hemisphere. The pre-Columbian T allele occurs at > 90% frequency within the native South and Central American populations examined, while its occurrence in North America is approximately 50%. Concomitant genotyping at the polymorphic tetranucleotide microsatellite DYS19 locus revealed that the C-->T mutation displayed significant linkage disequilibrium with the 186-bp allele. The data suggest a single origin of linguistically diverse native Americans with subsequent haplotype differentiation within radiating indigenous populations as well as post-Columbian European and African gene flow. The mutation may have originated either in North America at a very early time during the expansion or before it, in the ancestral population(s) from which all Americans may have originated. The analysis of linkage of the DYS199 and the DYS19 tetranucleotide loci suggests that the C-->T mutation may have occurred around 30,000 years ago. We estimate the nucleotide diversity over 4.2 kb of the nonrecombining portion of the Y chromosome to be 0.00014. compared to autosomes, the majority of variation is due to the smaller effective population size of the Y chromosome rather than selective sweeps. There begins to emerge a pattern of pronounced geographical localization of Y-specific nucleotide substitution polymorphisms.

Alleles↗