Non-syndromic neurosensory autosomal recessive deafness (NSRD) is the most common form of genetic hearing loss. Previous studies defined at least 15 human NSRD loci. Recently we demonstrated that DFNB1, located on the long arm of chromosome 13, accounts for approximately 80% of cases in the Mediterranean area. Further analysis with additional markers now identifies several recombinants which narrow the candidate region to approximately 5 cM, encompassed by markers D13S141 and D13S232 and including several ESTs and candidate genes, including the connexin26 (GJB2) gene. Analysis of PCR products from our affected patients' DNA shows two frameshift mutations in the connexin26 gene. Deletion of a G within a stretch of six Gs at position 35 of the GJB2 cDNA (mutation 35delG) leads to premature chain termination and is present in 63% of NSRD chromosomes, demonstrating linkage to chromosome 13. Deletion of a T at position 167 of GJB2 (mutation 167delT), also resulting in premature chain termination, was detected in another patient. Four neutral sequence polymorphisms were also identified. These findings are in agreement with a recent study showing that mutations in the connexin26 gene are associated with genetic forms of deafness in three Pakistani families and that GJB2 is DFNB1. Connexin26 is a member of a large family of proteins involved in formation of gap junctions, which are involved in electrical synapses and the direct transfer of small molecules and ionic currents between neighboring cells. The identification of GJB2 as the DFNB1 gene should provide a better understanding of the biology of normal and abnormal hearing, help form the basis for diagnosis and may facilitate development of strategies for treatment of this common genetic disorder.
We describe a rapid procedure for constructing cloned human genomic libraries from small amounts of peripheral blood. High molecular weight DNA is isolated from 5-20 ml peripheral blood, partially cleaved with Eco R1, and 8-22 kb fragments are cloned using bacteriophage Charon 4A and suitable E. coli host. Using the approach we have isolated and characterized several non-alpha globin clones from a Kurdish Jew with homozygous beta thalassemia. The ability to isolate suitable amounts of high molecular weight DNA from peripheral blood provides a relatively simple means of constructing human gene libraries representing a variety of hemoglobin disorders.
We describe a rapid "nonrandom" DNA sequence analysis procedure that facilitates the nucleotide sequence determination of large contiguous regions of DNA. The method consists of cloning a restriction endonuclease fragment of interest into bacteriophage M13 followed by construction of a series of nuclease BAL-31 deletion mutants originating from a single site in M13 that is close to the DNA insert. Determination of the size of the deletion mutant is accomplished by hybridization to a complementary single-stranded probe derived from M13 containing the total insert followed by nuclease SI treatment. Single-stranded M13-insert DNAs of progressively smaller sizes are isolated and analyzed by using a site-specific M13 DNA primer and the dideoxy chain-termination method. In this way, analysis of the DNA sequence proceeds from one -end of the total insert to the other in a nonrandom fashion due to generation ofa controlled overlapping set of deletion mutants.Advances in DNA sequence analysis techniques have revolutionized the study of cloned gene structure (1-3). The most commonly used method for DNA sequence analysis has been the chemical degradation procedure (1). There are several disadvantages to this technique because prior knowledge of the restriction endonuclease map of the fragment is required for detailed formulation of an analysis strategy. In addition, end labeling and sequence analysis from a single 5' or 3' end necessitates the use of relatively large amounts of purified fragment, a radioisotope of high specific activity, and relatively long exposure times for sequence readings.The use of bacteriophage M13 for subcloning and DNA sequence analysis offers distinct advantages in overcoming some of these difficulties. A number of recent reports describe the use of M13 for"random" DNA sequence analysis by the dideoxy chain-termination method using an M13 site-specific primer (4,5). This bacteriophage is well suited for DNA sequence analysis by the dideoxy chain-termination method, because cloning and isolation ofrecombinants is rapid and single-stranded phage DNA of he "+" strand covalently linked to single-stranded insert DNA is easily isolated from the culture medium. DNA sequence analysis is then accomplished by-the dideoxy chain-termination -method using the single-stranded M13 (+) insert DNA and a site-specific M13 primer that hybridizes close to the insert. One limitation of this technique is that only several hundred nucleotides from the primer site can be reliably read from gels. Therefore random analysis of a large DNA fragment involves the use of several restriction enzymes to develop a series ofsmall overlapping fragments that are subcloned into bacteriophage M13 and then analyzed. The entire DNA sequence is then assembled by matching of overlapping sequences, frequently with the aid of a computer. Another disadvantage of this random method is unnecessary redundancy in analysis; some regions may be analyzed several times before the entire DNA sequence can be assembled. Potential difficulties ...
The glycoprotein (GP) IIb/IIIa heterodimer functions as a receptor for fibrinogen, von Willebrand factor, and fibronectin on activated platelets; it is dysfunctional in the bleeding diathesis Glanzmann's thrombasthenia. This receptor is a member of the integrin family, which includes homologous membrane receptors involved in a number of different cell-cell and cell-matrix adhesive interactions. Knowledge of the sequence and organization of the GPIIb and GPIIIa genes will help in understanding evolutionary relationships and functional homologies of this family of adhesion protein receptors and will facilitate analysis of molecular defects responsible for thrombasthenia. Using the GPIIb cDNA as a probe, we have isolated overlapping genomic clones encompassing the entire coding region, the 5'- and 3'-untranslated sequences, and the immediate flanking regions for the GPIIb gene. The gene spans approximately 17.2 kilobases (kb); all but approximately 2.6 kb of intronic DNA sequence has been determined. The GPIIb gene contains 30 exons whose demarcations do not correlate with previously suggested functional domains. Two intron/exon borders have the rare GC splice donor sequence instead of the consensus GT sequence. There are at least seven complete and three partial AluI sequence repeats within the intron sequences. RNase protection, S1 nuclease analysis, and primer extension studies using human erythroleukemia (HEL) cell RNA and platelet RNA map a major transcription start site 32 base pairs (bp) 5' to the beginning of the coding region; however, there are no canonical consensus TATA or CAAT boxes in the region immediately 5' to the proposed cap site. The immediate 5'-flanking sequence of rodent GPIIb demonstrates complete identity near the proposed cap site with its human counterpart, but again, no TATA or CAAT boxes are apparent.
Deformable sickle erythrocytes have been reported by Mohandas and Evans to be more adherent to vascular endothelium than rigid irreversibly sickled cells (ISC). To define the clinical implications of this finding we have determined genetic, hematological, clinical, and rheological characteristics of sickle erythrocytes obtained from 65 patients with sickle cell anemia and fetal hemoglobin (Hb F) levels less than 15%. The alpha-globin gene number had a significant effect on the hematological parameters, the percentage of dense cells, ISC number, and HB A2 levels. The presence or absence of alpha thalassemia, however, had no effect on the frequency and severity of the sickle cell painful crisis (r = 0.06, P greater than .05). RBC deformability, determined by an ektacytometer, showed great heterogeneity among patients with three or four alpha-globin genes. Linear regression analyses of the data showed significant positive correlation of the frequency and severity of the painful crisis with RBC deformability (r = 0.49, P less than .001), and negative correlations with the percentage of dense cells (r = -0.37, P = .002), and the percentage of ISC (r = -0.46, P less than .001). We propose that the more deformable the sickle RBC are, the greater their adherence to vascular endothelium, and the more they cause vaso-occlusive crises, RBC deformability and the percentage of dense cells (or ISC) seem to have a predictive value of the frequency and severity of painful crises in sickle cell anemia.
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