RESUMEN
We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry delta F508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849 + 10kbC-->T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of delta F508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.
Asunto(s)
Fibrosis Quística/genética , Hispánicos o Latinos/genética , Proteínas de la Membrana/genética , Mutación Puntual , Secuencia de Aminoácidos , California , Cromosomas Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Genotipo , Haplotipos , Humanos , México/etnología , Sudoeste de Estados UnidosRESUMEN
We report DNA and clinical analyses of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene--namely, delta F508, G542X, G551D, R553X, N1303K, and W1282X--was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM19/PstI were performed as well. Of the 12 affected individuals studied, no delta F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. Our DNA data have serious implications for risk assessment of CF carrier status for these people.
Asunto(s)
Fibrosis Quística/genética , Genes Reguladores , Indígenas Norteamericanos/genética , Proteínas de la Membrana/genética , Adolescente , Adulto , Niño , Preescolar , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Análisis Mutacional de ADN , Exones , Femenino , Haplotipos/genética , Humanos , Lactante , Masculino , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Sudoeste de Estados UnidosRESUMEN
The Amylase locus in Drosophila melanogaster contains duplicate, divergently transcribed structural genes for alpha-amylase, AmyA and AmyB. A sensitive and reliable transient expression assay was developed for testing amylase activities produced by exogenous Amy genes in somatically transformed larvae of an amylase-null strain of flies. Alleles tested, AmyA and AmyB, came from recombinant clone lambda Dm65, which contains genomic DNA from a Canton-S strain. The transient assay was used in a deletion analysis aimed at locating cis-regulatory sequences within the 5' region of AmyB. Results suggest that upstream regulatory sequences for correct spatial expression of AmyA and AmyB in third-instar larvae are located within 446 and 430 bp of their respective starts for transcription. A sequence required for high levels of AmyB expression was located within its 5' upstream region between the base pairs at -332 and -219. AmyA does not appear to have a comparable regulatory element in its 5'-flanking sequence. Barely detectable expression of AmyB was observed when it was flanked by only 92 bp of upstream sequence. A model is proposed for incomplete coordinate control of the duplicate Amy genes.
Asunto(s)
Drosophila melanogaster/enzimología , Secuencias Reguladoras de Ácidos Nucleicos , alfa-Amilasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Deleción Cromosómica , Drosophila melanogaster/genética , Regulación Enzimológica de la Expresión Génica , Genes , Datos de Secuencia Molecular , Mutación , Plásmidos , Mapeo Restrictivo , Especificidad por Sustrato , alfa-Amilasas/metabolismoRESUMEN
The regulatory effects of allelic substitution at the trans-acting mapP locus and of dietary glucose on the expression of the duplicate genes for alpha-amylase (Amy) in Drosophila melanogaster were examined in the anterior midgut and posterior midgut regions of mature flies. The levels of amylase activity and amylase protein, as well as the abundance of amylase-specific RNA, were quantified. All 3 parameters of Amy expression were concordant. Results indicate that the effects of both mapP and dietary glucose are exerted at the level of amylase RNA. However, the tissue-specific effects of mapP are restricted to the posterior midgut and can therefore be distinguished from the effects of glucose in food medium, which influences amylase RNA levels in both the anterior and posterior midgut regions. Our data suggest that, in large part, strain-specific effects of dietary glucose can be explained on the basis of alternate alleles at the mapP locus in different homozygous strains of flies. Levels of amylase RNA in tissue extracts of flies from an amylase-null strain were also measured. Low levels were observed in both anterior and posterior midgut extracts. These were unresponsive to dietary conditions.
Asunto(s)
Dieta , Drosophila melanogaster/enzimología , Regulación Enzimológica de la Expresión Génica , alfa-Amilasas/genética , Animales , Sistema Digestivo/metabolismo , Drosophila melanogaster/genética , Femenino , Genes Reguladores , Glucosa/fisiología , Inmunoelectroforesis , Masculino , Fenotipo , ARN/metabolismoRESUMEN
The functional locus for alpha-amylase (Amy) in Drosophila miranda is in the evolutionarily new X2 chromosome. X2 evolved from an autosome in response to an ancestral autosome-Y translocation that gave rise to the "neo-Y" chromosome of this species. Y-linked Amy, if still present in the ancestrally translocated element, is unexpressed. Dosage compensation for amylase activity was examined in larvae of the S 204 strain. Since dietary glucose is known to repress Amy expression in Drosophila melanogaster, dosage compensation of amylase activity in male larvae of D. miranda was tested by rearing larvae of both sexes on yeast diets with or without a glucose supplement. The WT 10 strain of Drosophila persimilis, a sibling species in which Amy is autosomally linked, was used as a reference for tests of amylase activity differences between the sexes. On the diet with glucose, Amy expression was repressed in both WT 10 and S 204 larvae and male larvae of S 204 displayed dosage compensation for amylase activity. On the nonrepressing diet consisting of yeast alone, S 204 continued to display dosage compensation.
Asunto(s)
Compensación de Dosificación (Genética) , Drosophila/genética , Glucosa/metabolismo , alfa-Amilasas/genética , Animales , Evolución Biológica , Dieta , Drosophila/enzimología , Electroforesis en Gel de Poliacrilamida , Represión Enzimática , Femenino , Ligamiento Genético/genética , Isoenzimas , Larva , Masculino , Espectrofotometría , Translocación Genética , Cromosoma X , Cromosoma Y , alfa-Amilasas/biosíntesisRESUMEN
The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.
Asunto(s)
Amilasas/genética , Drosophila/genética , Isoenzimas/genética , Homología de Secuencia de Ácido Nucleico , Transformación Genética , Amilasas/biosíntesis , Animales , Clonación Molecular , Femenino , Regulación Enzimológica de la Expresión Génica , Plásmidos , Mapeo RestrictivoRESUMEN
Analysis of amylase RNA levels in the anterior and posterior midgut regions of flies from the Amy1,6 mapA and c Amy2,3 mapC strains of D. melanogaster, reared on yeast and on yeast supplemented with glucose, indicates that the trans-acting map gene controls the abundance of amylase RNA tissue-specifically, i.e., in the adult posterior midgut. This is consistent with the view that its role in controlling Amy expression is that of a transcription factor. Dietary glucose represses Amy expression in the anterior and posterior midgut regions of adults, reducing the abundance of amylase RNA, which suggests that it also controls Amy transcriptional activity. However, the mechanism for glucose repression appears to act systemically in the midgut, in a manner that is independent of the effects of map on Amy expression. A new glucose repressible TU was identified that is located just proximal to the Amy locus in region 54A of polytene chromosome 2R. It is transcribed in the direction opposite to that of the proximal Amy gene and encodes an RNA about 1500 bases long. Its RNA is expressed in both larvae and adults of the above strains of D. melanogaster, but the nature of the product it encodes is unknown. We speculate that all three genes in the cluster at 54A, namely the two Amy gene copies and the new glucose repressible TU, are coordinately controlled by the same mechanism that regulates Amy gene expression in response to dietary glucose. Somatic transformation experiments suggest that 5' cis-regulatory mechanisms required for the correct spatial expression of the proximal and distal Amylase genes from a Canton-S strain of D. melanogaster, Amy-p1 and Amy-d3, are located within 450 bp and 463 bp of their respective translation start sites. These regions also contain sequences responsive to dietary glucose repression, which is mediated at the DNA level of exogenous Amy genes in somatically transformed larvae reared on a yeast + glucose diet. A positive activator is located in the upstream region of Amy-d3 between the nucleotide pairs at -365 and -252 from the translation start site, but a comparable activator does not appear to exist in the upstream region of Amy-p1. Deletion analysis of the 5' sequence flanking the coding region of Amy-d3 indicates 125 nucleotide pairs of flanking DNA is sufficient for its functional activity. A model is proposed for coordinate control, in part, of the duplicated Amy genes.
Asunto(s)
Amilasas/genética , Drosophila melanogaster/genética , Familia de Multigenes , Animales , Drosophila melanogaster/enzimología , Genes Reguladores , Isoenzimas/genética , SeudogenesRESUMEN
Overlapping clones of the structural gene region for alpha-amylase, Amy, were isolated from a lambda EMBL4 library containing genomic DNA fragments from an amylase-null strain of Drosophila melanogaster. Southern blot analysis and restriction endonuclease mapping of the cloned region indicate that it contains an Amy gene duplication within an inverted repeat sequence as is characteristic of the genomic arrangement for this species. Spacing between the cloned gene copies is similar to that commonly found in other strains. Evidence is presented for the presence of an inversion 4 to 9 kb in length within the cloned Amy region of the null strain. We postulate a causal relationship between the presence of the inversion and the failure of individuals from the null strain to express amylase. A model is proposed that suggests the inversion may have arisen through intramolecular (or sister-strand) recombination mediated by homologous pairing of the inverted repeat sequences at the Amy locus.
Asunto(s)
Inversión Cromosómica , Clonación Molecular , Drosophila melanogaster/genética , Genes , alfa-Amilasas/genética , Animales , Southern Blotting , Drosophila melanogaster/enzimología , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
Chromosomal sites belonging to the alpha-amylase gene family have been identified in D. melanogaster and D. miranda and in the sibling species of miranda, pseudoobscura, and persimilis. Two sites occur in chromosome 2 of melanogaster; one contains the Amy gene locus (54A) and the other an amylase "pseudogene" (53CD). Two sites of homology exist at 73A and 78C and perhaps another at 81BC in chromosome 3 of pseudoobscura and persimilis and in the homologous regions of the X2 chromosome in miranda. The active Amy locus is apparently at 73A. The structural organization of cloned sequences from this multigene family in melanogaster and miranda is under analysis, with emphasis on the functional Amy gene region. Electrophoretic variants of amylase have served as invaluable tools in these studies. For melanogaster, their use as genetic markers enabled us to positively identify our lambda Dm65 clone of the Amy locus and to show that it contains two functional copies of the structural gene for alpha-amylase. Amylase isozymes are now being used in P element-mediated transformation experiments aimed at defining regulatory elements for the temporal and spatial control of amylase expression during development and in response to dietary glucose. In miranda, electrophoretic variants of amylase were useful in assigning the Amy locus to chromosome X2, and they continue to serve as essential markers in our study of the evolution of dosage compensation for amylase expression in males of this species. Restriction maps of the Amy locus in 7 strains of D. melanogaster indicate that despite the worldwide origins of the chromosome samples, all contain a duplication of the amylase structural gene at this locus regardless of whether they produce two alpha-amylase isozymes, a single variant, or none. We have aligned these maps with the genetic and cytological maps of chromosome 2R in melanogaster and assigned alleles for different amylase isozymes to either the proximal or distal Amy gene copy in a number of strains. Restriction site polymorphism is relatively limited at the Amy locus, but some strain-specific rearrangements exist. The locus of two strains with reduced amylase activity, Amy1 (CA 1) and Amy "null", contain anomalies--an insertion in the former and an inversion in the latter. Causal relationships are being sought between the level of amylase expression in these strains and the position of their respective anomalies.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Drosophila/genética , alfa-Amilasas/genética , Animales , Mapeo Cromosómico , Clonación Molecular , Drosophila/enzimología , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Isoenzimas/genética , Familia de Multigenes , Especificidad de la EspecieRESUMEN
A comparative developmental analysis was made of lipids from wild-type and adipose60 (adp60) mutants of Drosophila melanogaster. The lipid content and fatty acid profiles of late third instar larvae, pupae, and mature adults were characterized in methanol:chloroform extracts utilizing thin layer and gas-liquid chromatography. Total lipid content of mutant adults was approximately twice that of the wild-type, but no genotypic differences in lipid content were seen in earlier developmental stages. No sexual dimorphism was observed in total lipid content, although fatty acid profiles revealed some sexual differences. Many stage-specific differences in fatty acid profiles and lipid content were developmentally associated with each genotype. Mutants tended to retain the larval phenotype in lipid content and, to a lesser extent, in fatty acid profile. In comparison to wild-type, mutants tended to have increased lipid saturation, especially in 16-carbon fatty acids in mature adults and in 18:0 fatty acids in late larvae and pupae. No significant difference between the mutants and wild-type appeared in the developmental profiles for 14:1 fatty acid isomers. Hence, adp60 does not alter the desaturation-elongation pathway, a secondary pathway for fatty acid desaturation in Drosophila, which received support from this analysis.
Asunto(s)
Drosophila melanogaster/análisis , Ácidos Grasos/análisis , Lípidos/análisis , Animales , Cromatografía de Gases , Cromatografía en Capa Delgada , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Ácidos Grasos/genética , Femenino , Larva , Lípidos/genética , Masculino , Mutación , Pupa , Caracteres SexualesRESUMEN
Restriction maps were made by Southern blot analysis of the Amy (alpha-amylase) region in 7 strains of D. melanogaster using endonucleases SalI, XhoI and EcoRI. These were compared to the map of lambda Dm65 which contains the cloned Amy region. Strains used produce either two amylase variants, a single variant, or no amylase, yet all 7 strains carry two Amy genes as inverted repeats at the Amy locus. This and the orientation of the repeats resembles the situation in lambda Dm65. Most restriction sites mapped are conserved but two strains contain a large insertion which differs in size and position between strains. A complex anomaly, probably an inversion, exists at the Amy locus in a null strain. Maps for our Amy1,3 strain and the lambda Dm65 clone are identical, the DNA of each having been derived from a Canton-S wild stock. Restriction and genetic maps of the Amy region were aligned and alleles assigned to the proximal and distal genes, Amy-p and Amy-d.
Asunto(s)
Drosophila melanogaster/genética , alfa-Amilasas/genética , Animales , Deleción Cromosómica , Inversión Cromosómica , Clonación Molecular , Enzimas de Restricción del ADN , Isoenzimas/genética , Mutación , Especificidad de la EspecieRESUMEN
A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.
Asunto(s)
Clonación Molecular , Drosophila melanogaster/genética , Genes , alfa-Amilasas/genética , Animales , Mapeo Cromosómico , ADN/análisis , Drosophila melanogaster/enzimología , Ratones , Hibridación de Ácido Nucleico , Plásmidos , Saliva/enzimologíaRESUMEN
Restriction maps of an alpha-amylase structural gene clone, lambda Dm65, and of four putative alpha-amylase pseudogene clones are presented. Two alpha-amylase structural genes, inverted with respect to each other, are contained in lambda Dm65. Subregions of internal DNA sequence homology within lambda Dm65 and of cross-homology between the presumptive pseudogene clones and lambda Dm65 were determined. Subregions of cross-homology between the Drosophila clones and the mouse alpha-amylase cDNA clone, pMSa104, were also determined. The presence of functional alpha-amylase structural genes in lambda Dm65 was verified by injection of appropriate subclones into the germinal vesicle of Xenopus oocytes, followed by incubation of the oocytes under conditions that allowed coupled transcription and translation of injected genes to occur. Subclones of the 3.8- and 5.6-kb EcoRI fragments of lambda Dm65 were shown to code for alpha-amylase isozymes 1 and 3, respectively, of Drosophila melanogaster Canton-S. Both subclones are homologous to RNA of a size sufficient to accommodate the alpha-amylase-coding information. No RNA species homologous to other subcloned EcoRI fragments of lambda Dm65 was detected.
Asunto(s)
Clonación Molecular , Drosophila melanogaster/genética , Genes , Animales , Enzimas de Restricción del ADN , Drosophila melanogaster/enzimología , Femenino , Hibridación de Ácido Nucleico , Oocitos/metabolismo , Transcripción Genética , XenopusAsunto(s)
Drosophila melanogaster/genética , Genes , Isoenzimas/genética , alfa-Amilasas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Sistema Digestivo/enzimología , Drosophila melanogaster/enzimología , Femenino , Masculino , Especificidad de Órganos , Plásmidos , Biosíntesis de ProteínasRESUMEN
Laboratory strains of Drosophila melanogaster were screened for spatial variations in adult midgut alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) expression. No strain-specific differences were found anteriorly, but three patterns of activity were discerned in the posterior midgut: A, activity throughout most of the region; B, activity in the anterior part of the region; and C, little or no activity. Alleles of a control gene, map, are responsible for this tissue-specific regulation of activity; e.g., mapA homozygotes produce the A pattern and mapC homozygotes the C pattern. The map locus was placed at 2--80 +/- on the genetic map of chromosome 2R, about two crossover units distal to the Amy structural gene region for alpha-amylase. Electrophoretic studies showed that mapA is trans acting in mapA/mapC flies, allowing expression of amylase isozymes coded for by genes on the opposite chromosome. The map gene behaves as a temporal gene that is clearly separable from the tightly linked, duplicated Amy structural genes.