RESUMEN
AIM: Our goal wasto produce a field synopsis of genetic associations with preterm birth and to set up a publicly available online database summarizing the data. METHODS: We performed a systematic review and meta-analyses to identify genetic associations with preterm birth. We have set up a publicly available online database of genetic association data on preterm birth called PTBGene (http://ric.einstein.yu.edu/ptbgene/index.html) and report on a structured synopsis thereof as of December 1, 2008. RESULTS: Data on 189 polymorphisms in 84 genes have been included and 36 meta-analyses have been performed. Five gene variants (4 in maternal DNA, one in newborn DNA) have shown nominally significant associations, but all have weak epidemiological credibility. CONCLUSION: After publishing this field synopsis, the PTBGene database will be regularly updated to keep track of the evolving evidence base of genetic factors in preterm birth with the goal of promoting knowledge sharing and multicenter collaboration among preterm birth research groups.
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Genes/genética , Predisposición Genética a la Enfermedad , Bases del Conocimiento , Polimorfismo Genético/genética , Nacimiento Prematuro/genética , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Recién Nacido , Metaanálisis como Asunto , EmbarazoRESUMEN
We demonstrate the biocompatibility of carbon nanotube fibers (CNFs) fabricated from single-wall carbon nanotubes. Produced by a particle-coagulation spinning process, CNFs are "hair-like" conductive microwires, which uniquely combine properties of porous nanostructured scaffolds, high-area electrodes, and permeable microfluidic conduits. We report that CNFs are nontoxic and support the attachment, spreading, and growth of mammalian cells and the extension of processes from neurons in vitro. Our findings suggest that CNF may be employed for an electrical interfacing of nerve cells and external devices.
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Materiales Biocompatibles/administración & dosificación , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Nanotubos de Carbono , Neuronas/efectos de los fármacos , Neuronas/fisiología , Animales , Ensayo de Materiales , Ratones , Células 3T3 NIH , Neuronas/citología , Tamaño de la PartículaRESUMEN
Lactoferrin and lysozyme are important antimicrobial compounds of airway surface liquid, derived predominantly from serous cells of submucosal glands but also from surface epithelium. Here we compared release of these compounds from the following human cell cultures: primary cultures of tracheal epithelium (HTE), Calu-3 cells (a lung adenocarcinoma cell line frequently used as a model of serous gland cells), 16HBE14o- cells (an SV40 transformed line from airway surface epithelium), T84 cells (a colon carcinoma cell line), and human foreskin fibroblasts (HFF). For lysozyme, baseline secretory rates were in the order Calu-3 > 16HBE14o- > HTE T84 > HFF = 0; for lactoferrin, the only cell type showing measurable release was HTE; for mucus, HTE > Calu-3 > 16HBE14o- T84 > HFF = 0. A wide variety of neurohumoral agents and inflammatory stimuli was without effect on lactoferrin and lysozyme release from HTE or Calu-3 cells, although forskolin did stimulate secretion of water and lysozyme from Calu-3 cells. However, the concentration of lysozyme in the forskolin-induced secretions was much less than in airway gland secretions. Thus our data cast doubt on the utility of Calu-3 cells as a model of airway serous gland cells but do suggest that HTE could prove highly suitable for studies of mucin synthesis and release.
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Lactoferrina/metabolismo , Muramidasa/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Adenocarcinoma , Línea Celular Transformada , Línea Celular Tumoral , Fibroblastos/citología , Humanos , Neoplasias Pulmonares , Moco/metabolismo , Tráquea/citologíaRESUMEN
Virtually all in vitro studies of the effects of rhinovirus on human airway epithelium have used cells grown under conditions known to produce low levels of differentiation. The relevance of the results to native epithelium is questionable. Here we grew primary cultures of human tracheal or nasal epithelium under three conditions. One condition produced pseudostratified, mucociliary cells virtually indistinguishable from native epithelium. The other two conditions produced undifferentiated squamous cells lacking cilia. Cells were infected for 6 h with rhinovirus-16. After a 24-h incubation period, we determined levels of viral RNA in the cells, numbers of infectious viral particles released in the mucosal medium, expression of a variety of epithelial cytokines and other proteins, release of IL-6 and IL-8, and transepithelial electrical resistance and voltage. After infection, levels of viral RNA in the poorly differentiated cells were 30 or 130 times those in the differentiated. Furthermore, expression of mRNA for inflammatory cytokines, release of infectious particles, and release of IL-6 and IL-8 were closely correlated with the degree of viral infection. Thus well-differentiated cells are much more resistant to viral infection and its functional consequences than are poorly differentiated cells from the same source.
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Resfriado Común/inmunología , Células Epiteliales/virología , Mucosa Nasal/citología , Rhinovirus , Tráquea/citología , Diferenciación Celular , Células Cultivadas , Impedancia Eléctrica , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Inmunidad Innata , Interleucina-6/metabolismo , Interleucina-8/metabolismo , ARN Viral/análisis , Rhinovirus/genéticaRESUMEN
Sulfate conjugation is an important pathway in the metabolism of many drugs, xenobiotic compounds, and hormones. Sulfotransferases (SULTs) catalyze these reactions and have been detected and characterized in various human tissues including the liver and small intestine. Substrates for SULTs that include estrogen and thyroid hormones have well-established roles affecting skeletal integrity and disease processes. We performed the following studies to determine the presence of SULTs in human osteoblast-like cells, and to compare their characteristics to SULTs expressed in other human tissues. Four osteosarcoma cell lines (SaOS-2, U2-OS, PR, and HOS-TE85) were screened for the presence of four different SULT activities. Predominant activities were found for SULT1A1 in SaOS-2 cells, and SULT-1A3 in HOS-TE85 cells. Several biochemical properties of each enzyme that included apparent K(m) values, thermal stabilities, and responses to the inhibitors 2,6-dichloro-4-nitrophenol and NaCl were used to further characterize the SULT activities. High-performance liquid chromatography (HPLC) of the reaction products confirmed the known products of SULT1A1 and SULT1A3. When the mature human osteoblast HOB-03-CE6 cell line was tested for activity alone, the predominant activity was SULT1A3, with minimal SULT1A1. The results indicate that SULT1A1 and SULT1A3 are present in human osteosarcoma and mature osteoblast cell lines, and that the characteristics of the osteosarcoma cell SULTs are similar to those expressed in other human tissues. SULTs may have regulatory roles in the deactivation of thyroid hormones or estrogenic compounds in bone, and thus may affect hormone action and bone responses in the human skeleton.
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Arilsulfotransferasa , Osteoblastos/enzimología , Osteosarcoma/enzimología , Sulfotransferasas/metabolismo , Secuencia de Bases , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfotransferasas/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To determine the efficacy of combination therapy using atropine sulfate and albuterol in the treatment for an acute exacerbation of asthma. METHODS: A prospective, randomized double-blind, placebo-controlled study was performed in the ED of a large, inner-city, university-affiliated teaching hospital. Participants were a convenience sample of patients presenting to the ED between September 1993 and March 1994 with acute exacerbations of their asthma. Patients judged to be in extremis were excluded. All patients received 3 nebulized treatments with 2.5 mg of albuterol at 0, 30, and 60 minutes. Patients were randomized into 1 of 3 groups with the following added to their nebulizer solutions: 1) saline placebo during all 3 treatments; 2) 2.0 mg atropine sulfate added to the first nebulizer and saline in the second and third; or 3) 2.0 mg atropine to the first and third treatments (with saline in the second). No other medication was administered during the study period. At 90 minutes, the patients were evaluated for admission or release from the ED according to predetermined criteria, and additional medications were given as necessary. Vital signs, peak expiratory flow rate (PEFR), degree of wheezing, level of distress, and side effects were measured before and after each nebulizer treatment. RESULTS: Of the 153 patients eligible for the study, 126 completed the entire study protocol. There was no significant difference between the 3 groups on any parameter studied, including improvement of PEFR, vital signs, or level of distress. There was no difference in the admission rate between the 3 groups, nor was there a difference in the incidence of side effects among the groups. CONCLUSION: In this study population, combination therapy with atropine sulfate and albuterol offered no significant benefit over the use of albuterol alone in the treatment for acute exacerbation of asthma.
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Agonistas Adrenérgicos beta/uso terapéutico , Albuterol/uso terapéutico , Asma/tratamiento farmacológico , Atropina/uso terapéutico , Broncodilatadores/uso terapéutico , Antagonistas Colinérgicos/uso terapéutico , Adulto , Asma/fisiopatología , Atropina/farmacología , Presión Sanguínea/efectos de los fármacos , Método Doble Ciego , Quimioterapia Combinada , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Masculino , Ápice del Flujo Espiratorio/efectos de los fármacos , Estudios Prospectivos , Resultado del TratamientoRESUMEN
A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.
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Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , Rodopsina/biosíntesis , Opsinas de Bastones/biosíntesis , Animales , Western Blotting , Cloruro de Cadmio/farmacología , Bovinos , Células Cultivadas , Cromatografía de Afinidad , ADN Complementario/genética , Escherichia coli/genética , Genes Reporteros , Vectores Genéticos/genética , Humanos , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/genética , Retinaldehído/química , Rodopsina/genética , Opsinas de Bastones/genética , Sensibilidad y Especificidad , Transducina/metabolismo , Transfección , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Dental instruments and devices require sterilization or high-level disinfection. An evaluation of the implementation of such processes was undertaken. Eleven thousand questionnaires on methods used to sterilize and disinfect dental instruments were sent to dental practices and 1391 (13%) were returned for evaluation. Sixty-eight percent of respondents believed they were sterilizing their instruments, however, some of the liquid chemical products used were not suitable for sterilizing instruments, and 12% of respondents used incorrect contact times. Forty-nine percent of respondents did not challenge autoclaves with biological spores to check their function at an acceptable frequency. There were similar product and timing problems when a high-level liquid chemical disinfection was attempted. Although the return sample was small, problems were identified that can and should be corrected. This study demonstrates that the potential for person-to-person transmission of infectious agents such as the human immunodeficiency virus (HIV) and hepatitis B and C viruses via inadequately sterilized dental instrument exists depending on the prevalence of HIV in the dental practice area.
Asunto(s)
Equipo Dental , Instrumentos Dentales , Esterilización/métodos , Consultorios Odontológicos , Humanos , Mid-Atlantic Region , New England , Control de Calidad , Sudeste de Estados Unidos , Esterilización/normas , Encuestas y Cuestionarios , Factores de TiempoRESUMEN
The Sry gene functions as a genetic switch initiating testicular development of the indifferent mammalian gonad. The Mus musculus molossinus Sry open reading frame (ORF) encodes a 395-amino acid transcription factor (mSry) that specifically binds and bends DNA through its N-terminal HMG domain and activates transcription through its long C-terminal (residues 144-366) glutamine/histidine-rich activation domain. The M. m. domesticus Sry ORF encodes a highly homologous, truncated protein (dSry) of approximately 230 amino acids, and the molecular basis for truncation is a point mutation that creates an amber stop codon within the activation domain. The mSry protein activates transcription of a Sry-responsive reporter gene in HeLa cells, but dSry does not. Gene swapping and in vitro DNA binding experiments revealed that lack of transcriptional activation by dSry was not the result of polymorphisms within the first 137 amino acids of the protein. Direct analysis of the C-terminal glutamine/histidine-rich domain revealed that dSry lacked a functional transcriptional activation domain. Fusion of the GAL4 DNA-binding domain to the C-terminal deletion mutants of the GAL4-mSry chimeric protein indicated that residues 263-345 of the glutamine/histidine-rich domain were necessary for high level transactivation. Furthermore, readthrough of the premature amber stop codon by transfer RNA suppression resulted in a strong GAL4-dSry transactivator. This demonstrated that the premature stop codon is the only polymorphism responsible for the inability of the dSry glutamine/histidine-rich region to transactivate.
Asunto(s)
Proteínas de Unión al ADN/genética , Ratones/genética , Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Cricetinae , ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Reporteros , Células HeLa , Humanos , Sistemas de Lectura Abierta , Análisis para Determinación del Sexo , Proteína de la Región Y Determinante del Sexo , Activación Transcripcional , TransfecciónRESUMEN
Circular non-polyadenylated RNA molecules have been identified as stable transcription products of the human ETS-1 and mouse Sry genes. RNA circularization has been proposed to require two steps. The first step utilizes intramolecular base pairing to produce a transient stem-loop structure. The second step involves splicing a downstream donor splice site (DSS) to a now closely appositioned upstream acceptor splice site (ASS) within the loop. We demonstrate that the presence of long inverted repeats (IR) flanking the mouse Sry gene leads to the formation of the Sry circular transcript in cultured cells. Circularization requires the presence of both IR. As few as 400 complementary nt are necessary for this process. The presence of the IR does not significantly stimulate intermolecular annealing and trans-splicing in vivo.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Nucleares , ARN/química , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Transcripción , Animales , Secuencia de Bases , Cartilla de ADN/química , Expresión Génica , Ratones , Datos de Secuencia Molecular , Empalme del ARN , ARN Circular , ARN Mensajero/química , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Proteína de la Región Y Determinante del SexoRESUMEN
We have determined the complete nucleotide sequence (-865 to +3515) of the murine alpha B-crystallin/small heat shock protein gene, a major soluble protein of the vertebrate eye lens. Its 3 exon/2 intron structure is identical to that of the rat, hamster and human gene, with the exons being much more conserved than the introns. Previous reports indicated that there are two sizes of alpha B-crystallin mRNA; a larger alpha B-crystallin mRNA predominates in the lung and brain and is also found in low levels in most other tissues (except in lens and liver), while a smaller alpha B-crystallin mRNA exists at a high level in the lens and in variable amounts elsewhere. Sequence analysis suggests that secondary structure in the 5' untranslated sequence of the longer mRNA has led to difficulty in mapping the transcription initiation site of the longer transcript. Here we provide evidence by primer extension, S1 nuclease protection, and PCR (polymerase chain reaction) experiments for a transcription initiation site in the murine lung and brain at position -474. We also detected the utilization of the -474 initiation site in lens and of the +1 site in lung and brain, indicating that the tissue preference for these sites is not absolute. In vitro transcription experiments revealed that cell-free HeLa nuclear extracts specifically initiate transcription at the -474 and +1 sites. alpha B-crystallin was immunocytochemically localized to the bronchioles of the lung. Thus, regulation of alpha B-crystallin/small heat shock protein expression involves the utilization of tissue-preferred transcription initiation sites.
Asunto(s)
Cristalinas/genética , Proteínas de Choque Térmico/genética , Animales , Secuencia de Bases , Encéfalo/fisiología , Regulación de la Expresión Génica , Genes , Células HeLa , Humanos , Técnicas In Vitro , Intrones , Pulmón/fisiología , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Ratas , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination. The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal. The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity. When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a glutamine/histidine-rich domain. In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Nucleares , Factores de Transcripción/fisiología , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Núcleo Celular/química , Cricetinae , Proteínas de Unión al ADN/genética , Genes Reporteros , Genes Sintéticos , Humanos , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , Proteína de la Región Y Determinante del SexoRESUMEN
The alpha B-crystallin gene is expressed at high levels in lens and at lower levels in some other tissues, notably skeletal and cardiac muscle, kidney, lung, and brain. A promoter fragment of the murine alpha B-crystallin gene extending from positions -661 to +44 and linked to the bacterial chloramphenicol acetyltransferase (CAT) gene showed preferential expression in lens and skeletal muscle in transgenic mice. Transfection experiments revealed that a region between positions -426 and -257 is absolutely required for expression in C2C12 and G8 myotubes, while sequences downstream from position -115 appear to be determinants for lens expression. In association with a heterologous promoter, a -427 to -259 fragment functions as a strong enhancer in C2C12 myotubes and less efficiently in myoblasts and lens. Gel shift and methylation interference studies demonstrated that nuclear proteins from C2C12 myoblasts and myotubes specifically bind to the enhancer.
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Cristalinas/genética , Elementos de Facilitación Genéticos , Cristalino/metabolismo , Músculos/metabolismo , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Cristalinas/biosíntesis , ADN , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Plásmidos , Regiones Promotoras Genéticas , Conejos , TransfecciónRESUMEN
The crystallin genes encode the major soluble proteins of the lens. Some of the crystallin genes are expressed exclusively in the lens while others are also expressed in different tissues. The two alpha-crystallin genes, alpha A and alpha B, differ in their tissue specificity. Transcription of the alpha A-crystallin gene occurs only in the lens, while the alpha B-crystallin gene is also expressed in other tissues, including heart, skeletal muscle, kidney, lung and brain. MIP (also called MP26), the major intrinsic protein of the lens fiber membranes, is also expressed exclusively in the lens. Correct expression of both alpha-crystallin and MIP are required for normal lens function. Here we review our studies on the molecular basis of expression of the alpha-crystallin and MIP genes in the lens. The 5' flanking sequences containing the initiation site of transcription of the alpha A-crystallin, alpha B-crystallin and MIP genes were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the expression of this reporter gene was studied in transient assays and transgenic mice. DNA sequences flanking the 5' end of the alpha A-crystallin gene contain regulatory elements responsible for the lens-specific expression and developmental regulation of the CAT gene in transgenic mice. Interestingly, although some of the murine alpha A-crystallin regulatory sequences are conserved in the human and chicken genes, different functional regulatory elements appear to control the expression of the murine and chicken alpha A-crystallin genes. The 5' flanking sequence of the alpha B-crystallin gene preferentially directs expression of the CAT gene to the lens and to skeletal muscle. Different regulatory elements of the alpha B-crystallin gene appear to be responsible for its transcription in various tissues. The 5' flanking sequence of the MIP gene also contains regulatory elements that direct expression of the CAT gene to lens cells; these sequences are not functional in transfected non-lens cells and are different from the cis regulatory elements controlling alpha-crystallin gene expression. The multiplicity of cis-regulatory elements controlling the transcription of these three genes indicates the complexity of the mechanisms that regulate gene expression in the lens.
Asunto(s)
Cristalinas/genética , Proteínas del Ojo/genética , Glicoproteínas de Membrana , Animales , Acuaporinas , Secuencia de Bases , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de Secuencia , Transcripción GenéticaRESUMEN
alpha B-Crystallin, first identified as a structural component of the vertebrate eye lens, is expressed at high levels in lens and at lower levels in a number of other tissues, most notably cardiac and skeletal muscle, kidney, and brain. We have cloned and sequenced the human alpha B-crystallin gene and show that it is structurally similar to its hamster homolog. We have also identified its transcription initiation site in human lens RNA. Functional analysis of a promoter fragment extending from -537 to +21 (relative to the transcription initiation site) and fused to the bacterial chloramphenicol acetyltransferase gene suggests that this fragment contains regulatory elements that function preferentially, but not exclusively, in lens. In contrast, this fragment is apparently insufficient to promote transcription in glial cells, as this construct functioned poorly in a glioblastoma-astrocytoma cell line (U-373MG) that synthesizes high levels of the endogenous alpha B-crystallin gene product.
Asunto(s)
Cristalinas/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Genes , Humanos , Cristalino/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Using a human alpha B-crystallin genomic probe and human-mouse somatic cell hybrids, the human alpha B-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3-23.1.
Asunto(s)
Cromosomas Humanos Par 11 , Cristalinas/genética , Animales , Southern Blotting , Bandeo Cromosómico , Mapeo Cromosómico , Humanos , Células Híbridas , RatonesRESUMEN
Saccharomyces yeasts ferment several alpha-glucosides including maltose, maltotriose, turanose, alpha-methylglucoside, and melezitose. In the utilization of these sugars transport is the rate-limiting step. Several groups of investigators have described the characteristics of the maltose permease (D. E. Kroon and V. V. Koningsberger, Biochim. Biophys. Acta 204:590-609, 1970; R. Serrano, Eur. J. Biochem. 80:97-102, 1977). However, Saccharomyces contains multiple alpha-glucoside transport systems, and these studies have never been performed on a genetically defined strain shown to have only a single permease gene. In this study we isolated maltose-negative mutants in a MAL6 strain and, using a high-resolution mapping technique, we showed that one class of these mutants, the group A mutants, mapped to the MAL61 gene (a member of the MAL6 gene complex). An insertion into the N-terminal-coding region of MAL61 resulted in the constitutive production of MAL61 mRNA and rendered the maltose permease similarly constitutive. Transformation by high-copy-number plasmids containing the MAL61 gene also led to an increase in the maltose permease. A deletion-disruption of MAL61 completely abolished maltose transport activity. Taken together, these results prove that this strain has only a single maltose permease and that this permease is the product of the MAL61 gene. This permease is able to transport maltose and turanose but cannot transport maltotriose, alpha-methylglucoside, or melezitose. The construction of strains with only a single permease will allow us to identify other maltose-inducible transport systems by simple genetic tests and should lead to the identification and characterization of the multiple genes and gene products involved in alpha-glucoside transport in Saccharomyces yeasts.
Asunto(s)
Proteínas de Transporte de Membrana/genética , Saccharomyces/genética , Clonación Molecular , Genes Fúngicos , Genotipo , Cinética , Maltosa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Plásmidos , Mapeo Restrictivo , Saccharomyces/enzimología , Especificidad de la EspecieRESUMEN
The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.
Asunto(s)
Cristalinas/genética , Animales , Secuencia de Bases , Deleción Cromosómica , Clonación Molecular , Cristalinas/biosíntesis , ADN/genética , Regulación de la Expresión Génica , Genes Reguladores , Técnicas In Vitro , Cristalino/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Distribución TisularRESUMEN
Genetic analysis of the MAL6 locus has previously yielded mal6 mutants which fall into a single complementation group and which are noninducible for maltase and maltose permease. However, the strains used in these studies contained additional partially functional copies of MAL1 (referred to as MAL1g) and MAL3 (referred to as MAL3g). Using a strain lacking MALg genes, we have isolated two classes of mutants and these classes correspond to mutations in MAL63 and MAL61, two genes of the MAL6 complex. Disruptions of MAL63 are noninducible for maltase and maltose permease and for their corresponding mRNAs. The mal6 mutants are shown to map to MAL63. Inducer exclusion as a cause of the noninducible phenotype of the mal63 mutations has been eliminated by constructing a mal63 mutant in a strain constitutive for maltose permease; the strain remains noninducible. These results rigorously demonstrate that MAL63 is a regulatory gene which plays a positive role in the regulation of maltose fermentation.
Asunto(s)
Genes Fúngicos , Maltosa/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Mapeo Cromosómico , Inducción Enzimática , Fermentación , Prueba de Complementación Genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Monosacáridos , Mutación , Plásmidos , Mapeo Restrictivo , alfa-Glucosidasas/genéticaRESUMEN
Maltose fermentation in Saccharomyces carlsbergensis is dependent upon the MAL6 locus. This complex locus is composed of the MAL61 and MAL62 genes, which encode maltose permease and maltase, respectively, and a third gene, MAL63, which codes for a trans-acting positive regulatory product. In wild-type strains, expression of the MAL61 and MAL62 mRNAs and proteins is induced by maltose and induction is dependent upon the MAL63 gene. Mutants constitutively expressing the MAL61 and MAL62 gene products have been isolated in mal63 backgrounds, and the mutations which have been analyzed map to a fourth MAL6-linked gene, MAL64. Cloning and characterization of this new gene are described in this report. The results revealed that the MAL64-C alleles present in constitutive strains encode a trans-acting positive function required for constitutive expression of the MAL61 and MAL62 gene products. In inducible strains, the MAL64 gene is dispensable, as deletion of the gene had no effect on maltose fermentation or maltose-regulated induction. MAL64 encoded transcripts of 2.0 and 1.4 kilobase pairs. While both MAL64 mRNAs were constitutively expressed in constitutive strains, they were maltose inducible in wild-type strains and induction was dependent upon the MAL63 gene. The MAL63 and MAL64 genes are at least partially structurally homologous, suggesting that they control MAL61 and MAL62 transcript accumulation by similar mechanisms.