Isolation and characterisation of the NBR2 gene which lies head to head with the human BRCA1 gene.

To study the regulation of BRCA1 gene expression and the potential importance of dysregulation of this gene in breast and ovarian cancer, we have examined the 5' region of the human BRCA1 gene in detail. We have identified a new gene, NBR2, which is partially related to the NBR1 gene (formerly known as 1A1-3B and mapping directly adjacent to the pseudo-BRCA1 gene) and which lies head to head with the BRCA1 gene. The physical distance between the transcription start sites of the NBR2 and BRCA1 genes is 218 bp, suggesting that regulation of the expression of both genes may be co-ordinated through a bi-directional promoter. The NBR2 gene contains five exons spanning a genomic region of approximately 30 kb between the BRCA1 and pseudo-BRCA1 genes. Northern analysis showed that the NBR2 gene is expressed in all the tissues examined. The NBR2 cDNA contains an open reading frame of 112 amino acids and is predicted to encode a protein of approximately 12 kDa. Single-strand conformation polymorphism (SSCP) analysis of the NBR2 gene failed to identify any mutations in either breast or ovarian cancer, suggesting that if the NBR2 gene is involved in the development of these cancers, other mechanisms for tumorigenesis may exist. Hybridisation of NBR2 probes to zoo blots showed that the NBR2 gene is present in human and other primates. No hybridisation to DNA from other species was observed, suggesting that genomic elements controlling BRCA1 expression may differ between species.

Localization of the human RNA polymerase I transcription factor gene (UBTF) to the D17S183 locus on chromosome 17q21 and construction of a long-range restriction map of the region.

Human upstream binding factor (hUBF) is a sequence-specific DNA-binding protein that is essential for the activation of human 18s and 28s rRNA gene transcription. We have isolated and localized the gene (UBTF) encoding hUBF to the D17S183 locus on chromosome 17q21 by analyzing a cosmid from the region and carrying out Southern analysis on a previously constructed chromosome 17 somatic cell hybrid mapping panel using a probe from the hUBF cDNA. Confirmation of its location at this region was obtained from the results of pulsed-field gel electrophoresis analysis of genomic DNA using the hUBF cDNA and other probes from the region. These data also enabled the construction of a long-range restriction map of the region.

The SRY protein, like HMG 1, recognizes (CA)n sequences, an abundant repeat sequence in vertebrates.

The sex-determining region of the Y chromosome gene, sry is expressed in the foetal mouse for a brief period, just before testis differentiation, which could be consistent with negative autoregulation. SRY is a DNA binding protein which can bind to cruciform DNA and to linear DNA with a sequence specificity. We have examined if the Sry gene contain DNA binding sites for the SRY protein itself. We have found that in an in vitro assay, the SRY protein binds to several sites of the Sry gene and especially to a (CA)25 sequence and to a (CAG)30 repeat. These binding suggest that the function of SRY and in a general way HMG-box proteins may be mediated by an interaction with repeat sequences.

The detailed characterisation of a 400 kb cosmid walk in the BRCA1 region: identification and localisation of 10 genes including a dual-specificity phosphatase.

We have produced a detailed physical and transcriptional map of a 400 kb region within the narrowest flanking markers known to contain the hereditary breast and ovarian susceptibility gene, BRCA1. The approach described here has avoided the problems of chimaerism, instability and rearrangements commonly observed in yeast artificial chromosomes by converting the YAC clones into ordered chromosome 17-specific cosmid contigs and joining these contigs by cosmid end-walking. A detailed long-range restriction map provided a framework for the cosmid contig assembly and further refines existing physical mapping data. We have used a combined approach towards the isolation of the genes housed within these cosmids. This has resulted in the isolation and precise localisation of eight novel genes, including a novel G protein and an endogenous retrovirus related to the HERV-K family, and the previously described dual-specificity VHR phosphatase and MOX1 homeobox genes.

Genetic evidence equating SRY and the testis-determining factor.

The testis-determining factor gene (TDF) lies on the Y chromosome and is responsible for initiating male sex determination. SRY is a gene located in the sex-determining region of the human and mouse Y chromosomes and has many of the properties expected for TDF. Sex reversal in XY females results from the failure of the testis determination or differentiation pathways. Some XY females, with gonadal dysgenesis, have lost the sex-determining region from the Y chromosome by terminal exchange between the sex chromosomes or by other deletions. If SRY is TDF, it would be predicted that some sex-reversed XY females, without Y chromosome deletions, will have suffered mutations in SRY. We have tested human XY females and normal XY males for alterations in SRY using the single-strand conformation polymorphism assay and subsequent DNA sequencing. A de novo mutation was found in the SRY gene of one XY female: this mutation was not present in the patient's normal father and brother. A second variant was found in the SRY gene of another XY female, but in this case the normal father shared the same alteration. The variant in the second case may be fortuitously associated with, or predisposing towards sex reversal; the de novo mutation associated with sex reversal provides compelling evidence that SRY is required for male sex determination.

A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif.

A search of a 35-kilobase region of the human Y chromosome necessary for male sex determination has resulted in the identification of a new gene. This gene is conserved and Y-specific among a wide range of mammals, and encodes a testis-specific transcript. It shares homology with the mating-type protein, Mc, from the fission yeast Schizosaccharomyces pombe and a conserved DNA-binding motif present in the nuclear high-mobility-group proteins HMG1 and HMG2. This gene has been termed SRY (for sex-determining region Y) and proposed to be a candidate for the elusive testis-determining gene, TDF.

Structural analysis of water-soluble fractions obtained from Aspergillus fumigatus mycelium.

Fractions were prepared from the water-soluble components of Aspergillus fumigatus mycelium either by lectin-affinity chromatography or salt precipitation. While they varied considerably in their amino-acid composition, each contained a preponderance of aspartic and glutamic acids. 13C-NMR spectroscopy of these fractions, compared with that of polysaccharide obtained by alkaline extraction, indicated the presence of glycoproteins, the polysaccharide components of which contained beta-D-Galf units that are part of structures chemically different from those obtained by alkali treatment. In two of the three fractions examined, gas-liquid chromatography--mass spectrometry showed marked differences in the contents of non-reducing end-units of alpha-D-Manp and beta-D-Galf. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the preparations revealed an array of components, which stained to differing extents with silver stain and with Coomassie Blue and many of which were bound by lectins with specificity for different sugars.

Cornelia de Lange syndrome in several members of the same family.

A family is reported in which several members have the Cornelia de Lange syndrome and other members show facial dysmorphism and other features reminiscent of this syndrome. The segregation pattern is consistent with the view that the dysmorphic features (variable) are the manifestation of a single gene in heterozygous form. Chromosome abnormality was not found.

A study of erythrocyte membrane proteins and urinary polypeptides in conga drumming haemoglobinuria.

The erythrocyte membrane proteins and glycoproteins and urinary polypeptides have been examined in a patient exhibiting intermittent pigmenturia associated with conga drumming. Significant excretion of haemoglobin, albumin and probably erythrocyte carbonic anhydrase but not myoglobin occurred during the acute phase of the conga drumming-induced pigmenturia. This usually ceased within 24-48 h. We found no evidence of aberrant erythrocyte membrane components on electrophoresis with either protein staining or a range of 125I-labelled lectins used for detection.

Observations on the heparin neutralizing activity of outdated platelet concentrates.

A crude extract of outdated, washed, frozen and thawed, platelet concentrates was prepared and optimally diluted for use in the A.P.T.T. test as a source of platelet phospholipid (P.F. 3). The extract contained, in addition to P.F. 3, active heparin neutralizing activity (P.F. 4), which was able to neutralize plasma heparin up to a concentration of approximately 0.8 units/ml plasma. Although these observations are of a preliminary nature, it is felt that the extract can be used to follow the "intrinsic" coagulability of plasma samples from patients receiving therapeutic heparin. Utilizing the A.P.T.T. test system, the determination of coagulability and the neturalization of heparin result from the use of this extract in a single test.

A comparison of isometallothionein synthesis in rat liver after partial hepatectomy and parenteral zinc injection.

The time course of hepatic zinc-isometallothionein synthesis was studied in the regenerating liver and compared with that produced after the parenteral injection of zinc (6 mg of Zn2+/kg). In the regenerating liver, zinc levels rose rapidly after partial hepatectomy and reached a maximum at approx. 14h before declining to approximately normal levels at 48h post-operation. During this 48h period most of the zinc was incorporated into metallothionein. Purification of the latter into the charge-separable isometallothioneins (i.e. MT1 and MT2) showed that, in the regenerating liver, there was an unequal distribution of zinc between the two isoproteins. Thus at operation the endogenous thionein had an MT2/MT1 ratio of 1; after regeneration this ratio increased, and all times during the time course there was more MT2 than MT1. In contrast, the intraperitoneal injection of zinc produced a biphasic uptake of zinc into the liver with maxima at 10h and 32h. During the first phase of zinc uptake, metallothionein synthesis increased rapidly and, unlike the regenerating liver, the MT2/MT1 ratio of 1 remained constant. Thereafter, this ratio increased in a manner analogous to that exhibited by the regenerating liver. Half-life determinations for thionein disappearance/degradation shows that MT2 and MT1 were degraded with half-lives (t1/2) of 26.18h and 16.44h respectively in the regenerating liver and 14.75h and 9.3h after zinc injection. Thus thionein disappearance/degradation in the regenerating liver was slower than that seen after zinc injection. However, in both situations MT2 was always removed at a slower rate than MT1. Calculation of the rates of thionein synthesis (assuming the above disappearance rates were constant throughout the time course) showed that, in the regenerating liver, the rate of MT2 synthesis was approximately twice that of MT1. This was not the case after zinc injection, where both isometallothioneins were synthesized in equal amounts. These results demonstrate that the rates of synthesis of MT2 and MT1 can be altered according to the metabolic status of the cell and suggest a specific role for MT2 during liver regeneration.

Blood coagulation and fibrinolysis in thyroid disease.

Fibrinolytic activity and factor VIII concentration were studied in 30 patients with moderate to minimal hypothyroidism and in 7 patients with hyperthyroidism. In the hypothyroid group, the results were related to serum thyroxine levels, HL-A phenotypes and thyroid autoantibody titres. As serum thyroxine decreased so did factor VIII concentration, however, euglobulin lysis time was correspondingly prolonged. Factor VIII appears to be the most sensitive among coagulation factors to the deterioration of thyroid function tests. There was a significant correlation between the reciprocal of thyroid antibody titres and fibrinolysis; however, there was no relationship between factor VIII concentration or fibrinolysis and a specific HL-A phenotype although the incidence of HL-A8 was increased in the group as a whole. Euglobulin lysis time was prolonged in 6 out of 7 patients with Graves' hyperthyroidism. Factor VIII was elevated in only 3 of these patients.