RESUMEN

A sample of 526 Y chromosomes representing six Middle Eastern populations (Ashkenazi, Sephardic, and Kurdish Jews from Israel; Muslim Kurds; Muslim Arabs from Israel and the Palestinian Authority Area; and Bedouin from the Negev) was analyzed for 13 binary polymorphisms and six microsatellite loci. The investigation of the genetic relationship among three Jewish communities revealed that Kurdish and Sephardic Jews were indistinguishable from one another, whereas both differed slightly, yet significantly, from Ashkenazi Jews. The differences among Ashkenazim may be a result of low-level gene flow from European populations and/or genetic drift during isolation. Admixture between Kurdish Jews and their former Muslim host population in Kurdistan appeared to be negligible. In comparison with data available from other relevant populations in the region, Jews were found to be more closely related to groups in the north of the Fertile Crescent (Kurds, Turks, and Armenians) than to their Arab neighbors. The two haplogroups Eu 9 and Eu 10 constitute a major part of the Y chromosome pool in the analyzed sample. Our data suggest that Eu 9 originated in the northern part, and Eu 10 in the southern part of the Fertile Crescent. Genetic dating yielded estimates of the expansion of both haplogroups that cover the Neolithic period in the region. Palestinian Arabs and Bedouin differed from the other Middle Eastern populations studied here, mainly in specific high-frequency Eu 10 haplotypes not found in the non-Arab groups. These chromosomes might have been introduced through migrations from the Arabian Peninsula during the last two millennia. The present study contributes to the elucidation of the complex demographic history that shaped the present-day genetic landscape in the region.

Asunto(s)

Pool de Genes , Judíos/genética , Filogenia , Cromosoma Y/genética , Alelos , Árabes/genética , Emigración e Inmigración , Europa Oriental/etnología , Frecuencia de los Genes/genética , Variación Genética/genética , Haplotipos/genética , Humanos , Medio Oriente/etnología , Polimorfismo Genético/genética , Población Blanca/genética

RESUMEN

Chitinase A (ChiA) from the bacterium Serratia marcescens is a hydrolytic enzyme, which cleaves beta-1,4-glycosidic bonds of the natural biopolymer chitin to generate di-N-acetyl-chitobiose. The refined structure of ChiA at 1.55 A shows that residue Asp313, which is located near the catalytic proton donor residue Glu315, is found in two alternative conformations of equal occupancy. In addition, the structures of the cocrystallized mutant proteins D313A, E315Q, Y390F, and D391A with octa- or hexa-N-acetyl-glucosamine have been refined at high resolution and the interactions with the substrate have been characterized. The obtained results clearly show that the active site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates the substrate in the vicinity of the scissile bond. Furthermore, the enzyme imposes a critical "chair" to "boat" conformational change on the sugar residue bound to the -1 subsite. According to our results, we suggest that residues Asp313 and Tyr390 along with Glu315 play a central role in the catalysis. We propose that after the protonation of the substrate glycosidic bond, Asp313 that interacts with Asp311 flips to its alternative position where it interacts with Glu315 thus forcing the substrate acetamido group of -1 sugar to rotate around the C2-N2 bond. As a result of these structural changes, the water molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido group is displaced to a position that allows the completion of hydrolysis. The presented results suggest a mechanism for ChiA that modifies the earlier proposed "substrate assisted" catalysis.

Asunto(s)

Quitinasas/química , Quitinasas/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Serratia marcescens/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Conformación de Carbohidratos , Secuencia de Carbohidratos , Catálisis , Clonación Molecular , Cristalografía por Rayos X/métodos , Escherichia coli , Isopropil Tiogalactósido/química , Isopropil Tiogalactósido/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Sensibilidad y Especificidad , Programas Informáticos , Especificidad por Sustrato

RESUMEN

We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force-extension measurements of lambda DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA-IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA-IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA-IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.

Asunto(s)

Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , ADN/química , Proteínas Bacterianas/genética , Bacteriófago lambda/genética , ADN Viral/química , Proteínas de Unión al ADN/genética , Elasticidad , Factores de Integración del Huésped , Mutagénesis Sitio-Dirigida

RESUMEN

The proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (sigma(32)) is highly unstable. Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP-dependent protease FtsH. Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH(1) is highly stable. To determine which regions of E. coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins. Truncation of N-terminal amino acids had no, and deletion of C-terminal amino acids only a minor effect on stability of RpoH. A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36-122) roughly comprising the sigma factor region 2. This is the first demonstration of an internal RpoH region being responsible for FtsH-mediated degradation.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Factor sigma , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteasas ATP-Dependientes , Secuencia de Aminoácidos , Bradyrhizobium/enzimología , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , beta-Galactosidasa/metabolismo

RESUMEN

Deviation from the stepwise mutation model (SMM) at specific human microsatellite loci has implications for population genetic and forensic investigations. In the present study, data on six Y chromosome-specific microsatellites were pooled for 455 paternally unrelated males from six Middle Eastern populations. All chromosomes were assigned to three haplogroups defined by six binary polymorphisms. Two of the microsatellite loci tested, DYS388 and DYS392, displayed marked haplogroup-specific differences in their allele variability. A bimodal distribution of short and long alleles was observed for DYS388 in haplogroup 1 and for DYS392 in haplogroups 1 and 2. Further investigation showed that the short/long alleles segregated almost completely between genealogically distinct haplogroups defined by additional binary markers. Thus, these two loci have a discriminatory power similar to a binary polymorphism. DYS388 was characterised by an extremely low mutation rate in haplogroups 2 and 3, as was DYS392 in haplogroup 3. Sequence analysis of the repeat regions at the two loci revealed no irregularities, indicating that the triplet expansion in these loci is not controlled by sequence variation at the repeat level. A high frequency of long DYS388 alleles has, so far, been found only in populations originating in the Middle East, suggesting that this microsatellite is useful as a region-specific marker.

Asunto(s)

Haplotipos/genética , Repeticiones de Microsatélite/genética , Cromosoma Y/genética , Alelos , ADN/química , ADN/genética , Frecuencia de los Genes , Variación Genética , Humanos , Masculino , Modelos Genéticos , Mutación , Análisis de Secuencia de ADN

Asunto(s)

Virus 40 de los Simios/genética , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Virología/métodos , Animales , Baculoviridae/genética , Baculoviridae/aislamiento & purificación , Secuencia de Bases , Cápside/biosíntesis , Cápside/genética , Cápside/aislamiento & purificación , Línea Celular , ADN Viral/genética , Expresión Génica , Vectores Genéticos , Genoma Viral , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Spodoptera , Transfección , Ensayo de Placa Viral

RESUMEN

We introduce a method for detecting and tracking small particles in a solution near a surface. The method is based on blocking the backreflected illumination beam in an objective-type total internal reflection microscope, leaving unhindered the light scattered by the particles and resulting in dark-field illumination. Using this method, we tracked the motion of 60-nm polystyrene beads with a signal-to-noise ratio of 6 and detected 20-nm gold particles with a signal-to-noise ratio of 5. We illustrate the method's use by following the Brownian motion of small beads attached by short DNA tethers to a substrate.

RESUMEN

Linguistic evidence suggests that West Asia and Central Asia have been the two major geographical sources of genes in the contemporary Indian gene pool. To test the nature and extent of similarities in the gene pools of these regions we have collected DNA samples from four ethnic populations of northern India, and have screened these samples for a set of 18 Y-chromosome polymorphic markers (12 unique event polymorphisms and six short tandem repeats). These data from Indian populations have been analysed in conjunction with published data from several West Asian and Central Asian populations. Our analyses have revealed traces of population movement from Central Asia and West Asia into India. Two haplogrops, HG-3 and HG-9, which are known to have arisen in the Central Asian region, are found in reasonably high frequencies (41.7% and 14.3% respectively) in the study populations. The ages estimated for these two haplogroups are less in the Indian populations than those estimated from data on Middle Eastern populations. A neighbour-joining tree based on Y-haplogroup frequencies shows that the North Indians are genetically placed between the West Asian and Central Asian populations. This is consistent with gene flow from West Asia and Central Asia into India.

Asunto(s)

Genética de Población , Polimorfismo Genético , Cromosoma Y , Alelos , Asia Central , Asia Occidental , Evolución Molecular , Frecuencia de los Genes , Pool de Genes , Marcadores Genéticos , Variación Genética , Haplotipos , Humanos , India/etnología , Masculino , Dinámica Poblacional , Sensibilidad y Especificidad , Secuencias Repetidas en Tándem

RESUMEN

Alpha-thalassemia is among the world's most common single gene disorders, caused primarily by gene deletions. In Israel, where alpha(o)-trait thalassemia is uncommon, it is of particular importance because of its phenotypic interactions with beta-thalassemia in hetero- and homozygotes. In a study of 232 individuals referred for molecular evaluation of anemia, 303 chromosomes carried alpha-globin gene abnormalities; 6 gene rearrangements and 11 point mutations were identified. This unexpected heterogeneity is in part due to the many ethnic subgroups represented by these patients. Our findings include nine unique Israeli alleles, 3 of which are described here for the first time. An equal number of point mutations was found in the alpha2-globin gene as compared to alpha1. A threonine deletion in codon 39 of the alpha1-globin gene, found frequently in Arabs, is unique to Israel and probably represents one of several indigenous alleles. Among Arabs, point mutations were more frequent than large deletions. Surprisingly, in Ashkenazi Jews, who resided for many centuries in a nonmalarial environment, a single alpha-globin gene deletion -alpha(3.7) was found in many cases. The clinical presentation of individuals carrying two or more alpha-globin lesions was highly variable. In general, the severity correlated inversely with the number of functional alpha-globin genes. In some cases, impairment of two alpha-globin genes by point mutations led to a thalassemia-intermedia-like picture which could be misdiagnosed as beta-thalassemia. We conclude that alpha-thalassemia is phenotypically and genotypically more heterogeneous than previously recognized. DNA analysis is invaluable as it provides a specific diagnosis and enables reliable genetic counseling.

Asunto(s)

Globinas/genética , Talasemia alfa/genética , Etnicidad , Variación Genética , Genotipo , Humanos , Israel/epidemiología , Fenotipo , Mutación Puntual , Talasemia alfa/epidemiología

RESUMEN

Founder analysis is a method for analysis of nonrecombining DNA sequence data, with the aim of identification and dating of migrations into new territory. The method picks out founder sequence types in potential source populations and dates lineage clusters deriving from them in the settlement zone of interest. Here, using mtDNA, we apply the approach to the colonization of Europe, to estimate the proportion of modern lineages whose ancestors arrived during each major phase of settlement. To estimate the Palaeolithic and Neolithic contributions to European mtDNA diversity more accurately than was previously achievable, we have now extended the Near Eastern, European, and northern-Caucasus databases to 1,234, 2, 804, and 208 samples, respectively. Both back-migration into the source population and recurrent mutation in the source and derived populations represent major obstacles to this approach. We have developed phylogenetic criteria to take account of both these factors, and we suggest a way to account for multiple dispersals of common sequence types. We conclude that (i) there has been substantial back-migration into the Near East, (ii) the majority of extant mtDNA lineages entered Europe in several waves during the Upper Palaeolithic, (iii) there was a founder effect or bottleneck associated with the Last Glacial Maximum, 20,000 years ago, from which derives the largest fraction of surviving lineages, and (iv) the immigrant Neolithic component is likely to comprise less than one-quarter of the mtDNA pool of modern Europeans.

Asunto(s)

ADN Mitocondrial/genética , Efecto Fundador , Pool de Genes , Filogenia , Bases de Datos como Asunto , Emigración e Inmigración , Europa (Continente) , Herencia Extracromosómica/genética , Frecuencia de los Genes/genética , Variación Genética/genética , Haplotipos/genética , Humanos , Medio Oriente/etnología , Mutagénesis , Factores de Tiempo

RESUMEN

The Escherichia coli heat shock sigma factor sigma32 (RpoH) is rapidly degraded under non-stress conditions. The integrity of the DnaK chaperone machinery and the ATP-dependent FtsH protease are required for sigma32 proteolysis in vivo. Bradyrhizobium japonicum expresses three sigma32-type transcription factors, RpoH1, RpoH2, and RpoH3, which are functional in E. coli. We compared the stability of these sigma factors with E. coli sigma32 stability. In E. coli C600 (wild-type), the half-lives of sigma32, RpoH1, RpoH2 and RpoH3 were 30 s, 7 min, 4 min and 4 min, respectively. The first three proteins were stabilized in ftsH mutant backgrounds, indicating that they are degraded by FtsH in the wild-type. Proteolysis of RpoH3 was FtsH-independent because this sigma factor was not stabilized in ftsH mutants. Interestingly, in a purified in vitro system, all four RpoH proteins were degraded by FtsH, indicating that in vivo protein degradation depends on additional cellular factors. Rationally designed point mutations of sigma32 and RpoH1 suggested that the highly conserved RpoH box does not play a major role in conferring stability to RpoH factors. Presumably, several regions distributed along the primary sequence of the sigma factor are important for FtsH-mediated proteolysis. Finally, we provide evidence that proteolysis of RpoH factors in vivo depends on the DnaK machinery, irrespective of the protease involved.

Asunto(s)

Bradyrhizobium/química , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Choque Térmico/metabolismo , Factor sigma , Factores de Transcripción/metabolismo , Proteasas ATP-Dependientes , Proteínas Bacterianas/metabolismo , Bradyrhizobium/metabolismo , Cloranfenicol/farmacología , Escherichia coli/metabolismo , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Calor , Immunoblotting , Isopropil Tiogalactósido/farmacología , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Péptidos/inmunología , Péptidos/metabolismo , Mutación Puntual , Proteínas Recombinantes/metabolismo , Temperatura , Factores de Tiempo , Factores de Transcripción/genética

RESUMEN

The catalytic domain of chitobiase (beta-N-1-4 acetylhexosaminidase) from Serratia marcescens, is an alpha/beta TIM-barrel. This enzyme belongs to family 20 of glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp539-Glu540). It was proposed that catalysis by this enzyme family is carried out by glutamate 540 acting as a proton donor and by the acetamido group of the substrate as a nucleophile. We investigated the role of Asp539 and Glu540 by site-directed mutagenesis, biochemical characterization and by structural analyses of chitobiase -substrate co-crystals. We found that both residues are essential for chitobiase activity. The mutations, however, led to subtle changes in the catalytic site. Our results support the model that Glu540 acts as the proton donor and that Asp539 acts in several different ways. Asp539 restrains the acetamido group of the substrate in a specific orientation by forming a hydrogen bond with N2 of the non-reduced (-1) sugar. In addition, this residue participates in substrate binding. It is also required for the correct positioning of Glu540 and may provide additional negative charge at the active site. Thus, these biochemical and structural studies provide a molecular explanation for the functional importance and conservation of these residues.

Asunto(s)

Acetilglucosamina/metabolismo , Acetilglucosaminidasa/química , Acetilglucosaminidasa/metabolismo , Ácido Aspártico/metabolismo , Ácido Glutámico/metabolismo , Mutación/genética , Serratia marcescens/enzimología , Acetilglucosamina/análogos & derivados , Acetilglucosaminidasa/genética , Sustitución de Aminoácidos , Ácido Aspártico/genética , Sitios de Unión , Catálisis , Secuencia Conservada/genética , Cristalización , Cristalografía por Rayos X , Ácido Glutámico/genética , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Serratia marcescens/genética , Relación Estructura-Actividad , Termodinámica

RESUMEN

The molecular basis of the thalassemias has been studied in many of the world's populations. Here we report the results of the first screening for mutations in Vietnam. Twenty-three unrelated patients, of which 17 have Hb E/beta-thalassemia, were diagnosed and beta-globin mutations were detected in all 46 chromosomes. Four previously reported South Asian mutations were found. The most common mutations were the nonsense in codon 17 (A-->T) and the frameshift at codons 41/42 (-TTCT) (30 and 22%, respectively). The rare frameshift mutation at codon 95 (+A) was present in 9% of the 46 chromosomes studied, suggesting that it is indigenous to Vietnam. These results will serve as an initial database for DNA-based prenatal diagnosis of thalassemia in Vietnam.

Asunto(s)

Talasemia beta/genética , Niño , Preescolar , Mutación del Sistema de Lectura , Frecuencia de los Genes , Pruebas Genéticas , Hemoglobina E/genética , Humanos , Mutación , Mutación Missense , Sondas de Oligonucleótidos , Vietnam/epidemiología

RESUMEN

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).

Asunto(s)

Proteínas Bacterianas/química , Escherichia coli/enzimología , Proteínas de la Membrana/química , Factor sigma , Proteasas ATP-Dependientes , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Secuencia Conservada/genética , Reactivos de Enlaces Cruzados/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Factores de Transcripción/metabolismo , Tripsina/metabolismo , Proteínas Virales

RESUMEN

FtsH (HflB) is a conserved, highly specific, ATP-dependent protease for which a number of substrates are known. The enzyme participates in the phage lambda lysis-lysogeny decision by degrading the lambda CII transcriptional activator and by its response to inhibition by the lambda CIII gene product. In order to gain further insight into the mechanism of the enzymatic activity of FtsH (HflB), we identified the peptides generated following proteolysis of the phage lambda CII protein. It was found that FtsH (HflB) acts as an endopeptidase degrading CII into small peptides with limited amino acid specificity at the cleavage site. beta-Casein, an unstructured substrate, is also degraded by FtsH (HflB), suggesting that protein structure may play a minor role in determining the products of proteolysis. The majority of the peptides produced were 13 to 20 residues long.

Asunto(s)

Proteínas Bacterianas/metabolismo , Bacteriófago lambda , Endopeptidasas/metabolismo , Escherichia coli/enzimología , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Factores de Transcripción/metabolismo , Proteasas ATP-Dependientes , Secuencia de Aminoácidos , Caseínas/metabolismo , Proteínas de Escherichia coli , Datos de Secuencia Molecular , Especificidad por Sustrato , Factores de Transcripción/aislamiento & purificación , Proteínas Virales

RESUMEN

Haplotypes constructed from Y-chromosome markers were used to trace the paternal origins of the Jewish Diaspora. A set of 18 biallelic polymorphisms was genotyped in 1,371 males from 29 populations, including 7 Jewish (Ashkenazi, Roman, North African, Kurdish, Near Eastern, Yemenite, and Ethiopian) and 16 non-Jewish groups from similar geographic locations. The Jewish populations were characterized by a diverse set of 13 haplotypes that were also present in non-Jewish populations from Africa, Asia, and Europe. A series of analyses was performed to address whether modern Jewish Y-chromosome diversity derives mainly from a common Middle Eastern source population or from admixture with neighboring non-Jewish populations during and after the Diaspora. Despite their long-term residence in different countries and isolation from one another, most Jewish populations were not significantly different from one another at the genetic level. Admixture estimates suggested low levels of European Y-chromosome gene flow into Ashkenazi and Roman Jewish communities. A multidimensional scaling plot placed six of the seven Jewish populations in a relatively tight cluster that was interspersed with Middle Eastern non-Jewish populations, including Palestinians and Syrians. Pairwise differentiation tests further indicated that these Jewish and Middle Eastern non-Jewish populations were not statistically different. The results support the hypothesis that the paternal gene pools of Jewish communities from Europe, North Africa, and the Middle East descended from a common Middle Eastern ancestral population, and suggest that most Jewish communities have remained relatively isolated from neighboring non-Jewish communities during and after the Diaspora.

Asunto(s)

Pool de Genes , Haplotipos , Judíos/genética , Cromosoma Y , Secuencia de Bases , Evolución Biológica , Humanos , Masculino , Datos de Secuencia Molecular

RESUMEN

The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate-functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

Asunto(s)

Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Tilacoides/enzimología , Proteasas ATP-Dependientes , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Proteínas de Arabidopsis , Caseínas/metabolismo , Dominio Catalítico , Cationes/farmacología , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Luz , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Metaloendopeptidasas/genética , Metaloendopeptidasas/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Complejo de Proteína del Fotosistema II , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Tilacoides/efectos de los fármacos

RESUMEN

An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB). These mutations were demonstrated to reduce the ability of FtsH to degrade the phage lambda CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype.

Asunto(s)

Proteínas Bacterianas/genética , Colicinas/farmacología , Escherichia coli/genética , Proteínas de la Membrana/genética , Mutación , Proteasas ATP-Dependientes , Proteínas Bacterianas/metabolismo , Bacteriófago lambda/fisiología , Clonación Molecular , Escherichia coli/efectos de los fármacos , Escherichia coli/virología , Proteínas de Escherichia coli , Regulación Viral de la Expresión Génica , Lisogenia/fisiología , Proteínas de la Membrana/metabolismo , Fenotipo , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo , Transducción Genética , Proteínas Virales

RESUMEN

The potential and reliability of DNA analysis for the identification of human remains are demonstrated by the study of a recent bone sample, which represented a documented case of sickle cell anemia. beta-globin gene sequences obtained from the specimen revealed homozygosity for the sickle cell mutation, proving the authenticity of the retrieved residual DNA. Further investigation of mitochondrial and Y chromosome DNA polymorphic markers indicated that this sample came from a male of maternal West African (possibly Yoruban) and paternal Bantu lineages. The medical record, which became available after the DNA analyses had been completed, revealed that it belonged to a Jamaican black male. These findings are consistent with this individual being a descendent of Africans brought to Jamaica during the trans-Atlantic slave trade. This study exemplifies how a "reverse population genetics" approach can be applied to reconstruct a genetic profile from a bone specimen of an unknown individual.

Asunto(s)

Anemia de Células Falciformes/genética , Dermatoglifia del ADN , Genética de Población , África , Antropología Física , ADN Mitocondrial/genética , Humanos , Masculino , Cromosoma Y/genética

RESUMEN

Production of the major capsid protein of SV40, VP1, is of great interest for the study on capsid assembly in vitro. Production of soluble His6-VP1 in Escherichia coli strains deficient in the GroELS chaperone machine was substantially higher than in the wild-type strain. The His6-VP1 produced in a groEL mutant strain was readily purified. The protein was able to form higher-order structures as evidenced by analysis of the soluble fraction by gel filtration, by sedimentation in sucrose gradient, and by electron microscopy. We propose the use of groE mutants for the production of the major capsid protein of SV40 and perhaps also other papovaviruses.

Asunto(s)

Proteínas de la Cápside , Cápside/biosíntesis , Cápside/aislamiento & purificación , Chaperonina 10/genética , Chaperonina 60/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Poliomavirus/genética , Cápside/genética , Chaperonina 10/biosíntesis , Chaperonina 10/metabolismo , Chaperonina 60/biosíntesis , Chaperonina 60/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Líquido Intracelular/metabolismo , Virión/genética , Virión/metabolismo