Igor Medintz

Metabolic Signals Trigger Glucose-Induced Inactivation of Maltose Permease in Saccharomyces

Organisms such as Saccharomyces capable of utilizing several different sugars selectively ferment glucose when less desirable carbon sources are also available. This is achieved by several mechanisms. Glucose down-regulates the transcription of genes involved in utilization of these alternate carbon sources. Additionally, it causes posttranslational modifications of enzymes and transporters, leading to their inactivation and/or degradation. Two glucose sensing and signaling pathways stimulate glucose-induced inactivation of maltose permease. Pathway 1 uses Rgt2p as a sensor of extracellular glucose and causes degradation of maltose permease protein. Pathway 2 is dependent on glucose transport and stimulates degradation of permease protein and very rapid inactivation of maltose transport activity, more rapid than can be explained by loss of protein alone. In this report, we characterize signal generation through pathway 2 using the rapid inactivation of maltose transport activity as an assay of signaling activity. We find that pathway 2 is dependent on HXK2 and to a lesser extent HXK1. The correlation between pathway 2 signaling and glucose repression suggests that these pathways share common upstream components. We demonstrate that glucose transport via galactose permease is able to stimulate pathway 2. Moreover, rapid transport and fermentation of a number of fermentable sugars (including galactose and maltose, not just glucose) are sufficient to generate a pathway 2 signal. These results indicate that pathway 2 responds to a high rate of sugar fermentation and monitors an intracellular metabolic signal. Production of this signal is not specific to glucose, glucose catabolism, glucose transport by the Hxt transporters, or glucose phosphorylation by hexokinase 1 or 2. Similarities between this yeast glucose sensing pathway and glucose sensing mechanisms in mammalian cells are discussed.

Intracellular Maltose Is Sufficient To Induce MAL Gene Expression in Saccharomyces cerevisiae

ABSTRACT The presence of maltose induces MAL gene expression in Saccharomyces cells, but little is known about how maltose is sensed. Strains with all maltose permease genes deleted are unable to induce MAL gene expression. In this study, we examined the role of maltose permease in maltose sensing by substituting a heterologous transporter for the native maltose permease. PmSUC2 encodes a sucrose transporter from the dicot plant Plantago major that exhibits no significant sequence homology to maltose permease. When expressed in Saccharomyces cerevisiae, PmSUC2 is capable of transporting maltose, albeit at a reduced rate. We showed that introduction of PmSUC2 restores maltose-inducible MAL gene expression to a maltose permease-null mutant and that this induction requires the MAL activator. These data indicate that intracellular maltose is sufficient to induce MAL gene expression independently of the mechanism of maltose transport. By using strains expressing defective mal61 mutant alleles, we demonstrated a correlation between the rate of maltose transport and the level of the induction, which is particularly evident in medium containing very limiting concentrations of maltose. Moreover, our results indicate that a rather low concentration of intracellular maltose is needed to trigger MAL gene expression. We also showed that constitutive overexpression of either MAL61 maltose permease or PmSUC2 suppresses the noninducible phenotype of a defective mal13 MAL-activator allele, suggesting that this suppression is solely a function of maltose transport activity and is not specific to the sequence of the permease. Our studies indicate that maltose permease does not function as the maltose sensor in S. cerevisiae.

Restriction Fragment Length Polymorphism and Polymerase Chain Reaction-HLA DQα Analysis of Casework Urine Specimens

DNA was isolated from casework urine samples previously submitted for toxicological analysis. The quality and quantity of DNA isolated was determined by spectrofluorometry and agarose yield gel electrophoresis. Hae III restricted samples were then resolved by analytical agarose gel electrophoresis, transferred to a membrane by Southern blotting and hybridized with a chemiluminescently-labelled (D2S44) probe. The DNA fragment banding patterns were indistinguishable from the DNA banding patterns of blood specimens collected from the same donor. Only 5 of 20 samples yielded banding patterns and the banding intensity relative to background was low. Genomic DNA was also obtained from casework samples by Chelex extraction, amplified by polymerase chain reaction (PCR) and then genotyped for human leucocyte antigen (HLA) DQ alpha. Of 20 specimens, 13 (65%) were typed correctly producing identical results for urine and blood specimens obtained from the same donor. Aging studies of casework samples and normal samples (from a non-drug using population) were also conducted with PCR-HLA DQ alpha analysis. Results of these studies indicate that amplification by PCR was more likely to produce positive results. Based on these findings, we conclude that PCR-initiated analysis is more suitable than RFLP analysis for individualization of urine samples.

HLA-DQA1 and polymarker allele frequencies in two New York City Jewish populations.

Allele and genotype frequencies were determined for the HLA-DQA1 and Amplitype Polymarker loci (low density lipoprotein receptor (LDLR), glycophorin A (GYPA), hemoglobin G gammaglobin (HBGG), D7S8, and group-specific component (Gc)) in Hasidic and non-Hasidic Ashkenazi New York City Jewish subpopulations. For all loci tested, except HBGG, the 2 subpopulations meet the assumption of Hardy-Weinberg equilibrium. Comparison of various allele and genotype frequencies for the Hasidic and the non-Hasidic groups showed no significant differences. Comparison of the various allele frequencies in the two subpopulations to another Caucasian group revealed significant differences at the HLA-DQA1 and D7S8 loci in the Hasidic group. These frequency data can be used for comparison to other populations and for frequency estimates in DNA profiling.

DlS80 allele frequencies in Hasidic and non-Hasidic New York City Jewish populations

Abstract Allele frequencies were determined for the VNTR locus D1S80 in Hasidic and non-Hasidic Ashkenazi New York City Jewish subpopulations. Samples were amplified via the polymerase chain reaction and underwent genotyping using polyacrylamide gel electrophoresis. In the Hasidic population 14 alleles were observed as opposed to 19 alleles in the non-Hasidic community. Both populations were tested for Hardy-Weinberg equilibrium. The frequency data obtained can be used for comparison to other populations and for allele and genotype frequency estimates in genetic marker profiling of evidentiary specimens.

Determination of identity in paraffin-embedded tissues and slides

Abstract A procedure is presented for the determination of donor identity or commonality of origin among paraffinembedded tissues and slides. Samples are de-paraffinized with xylene and ethanol and subjected to ChelexR extraction. The Polymarker forensic DNA typing kit is utilized for identity testing. Following multiplex amplification, PCR product is verified on an agarose gel and then the samples are geiotyped using reverse dot-blot hybridization. When this procedure was applied to paraffin-embedded samples involved in a legal action, all samples were successfully genotyped. The advantages of using this procedure are discussed.

DNA Analysis of Urine Stained Material

Abstract Suitability of the DNA content in urine stained material was evaluated for use in forensic analysis. High molecular weight (HMW) DNA (>20 kb, where 1kb equals 1000 base pairs) was obtained in only 9% (4 out of 44) of laboratory produced urine stains using an organic extraction method. Slot blot hybridization analysis with a human specific repeat probe (SLI332) showed that these stains contained DNA of human origin. A group of 12 urine stains from the original 44 that did not yield any DNA was subjected to Chelex resin extraction followed by PCR amplification for genotyping of the Human Leucocyte Antigen (HLA) DQA1 locus. Typing was successfull on 5 of the 12 samples. The concentrated sediment from the original liquid urine portion of the samples was similarly subjected to Chelex extraction and HLA DQA1 typing. Six out of ten samples were successfully typed. Blood specimens collected from the urine donors to serve as typing controls yielded identical genotypes in all cases. PCR based analysis of u...