Kallikrein and prekallikrein levels in a large number of congenital clotting deficiencies and abnormalities

Kallicrein (K) and prekallicrein (PK) were assayed in a large number of cases with congenitial clotting factor defects. Patients with factor XII deficiency were separated from other clotting abnormalities. The results were compared with a control group of normal subjects. We found significantly reduced PK activity levels in the factor XII deficient group. Although less evident, the reduction of PK activity in the group of other clotting defects was modest, however, not due to a factor VII defect. In our study we found that in the absence of factor XII, PK is not activated. Further studies will be necessary to show if PK activation is altered or reduced in other congenital clotting abnormalities.     

Identification of growth hormone DNA polymorphisms which respond to divergent selection for abdominal fat content in chickens

Summary Two strains of meat-type chickens which had been derived from the same genetic base, but were selected for high or low abdominal fat content, respectively, were analyzed for polymorphisms in the growth hormone gene (GH). A total of four DNA polymorphisms were identified, one at a SacI restriction site and three at MspI restriction sites. Restriction mapping indicated that all polymorphisms were in exons and/or introns and not in flanking regions of the gene. The incidence of GH polymorphisms was determined in 20 chickens from each strain and significant differences were observed for two of the four polymorphisms. Analysis by DNA fingerprinting using (CAC)₅ as a probe indicated that the inbreeding coefficient was 0.1 in both strains and that random genetic drift was minimal. Thus, the selection for abdominal fat appears to have affected the frequency of alleles of the growth hormone gene. Whether this is the direct consequence of an altered growth hormone gene on fat metabolism or reflects linkage to an allele of a neighbouring gene remains to be determined.     

A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino-acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient’s brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal. Received: 16 December 1996 / Accepted: 5 May 1997     

Yeast Comparative Genetics: A New MEL15 α-Galactosidase Gene of Saccharomyces cerevisiae

To supplement the earlier identified European family of the highly homologous -galactosidase MEL1–MEL11genes and the African family of the divergent MEL12–MEL14 genes, a new MEL15gene was found in several Saccharomyces cerevisiae strains isolated from maize dough in Ghana. Southern blotting and restriction enzyme analysis assigned the MEL15 gene to the African family and mapped it to chromosomes IV/XII, which migrate together in electrophoresis. Tetrad analysis ruled out the MEL15 location in the left arm of chromosome IV or the right arm of chromosome XII, which respectively contain the known MEL5 and MEL10 genes.     

Expression of enhancing factor/phospholipase A2 in ICRC mice

Enhancing factor (EF), a growth factor modulator, recently identified as the mouse secretory phospholipase A₂ (PLA₂), has been isolated in our laboratory from the intestines of mice. EF modulates the action of epidermal growth factor (EGF) by mediating an almost 2-fold increase in EGF binding in a radioreceptor assay. EF has been localized immunohistochemically to the Paneth cells of the intestine, adjacent to the proliferating stem cell population. Although very weak staining was observed in the intestines of ICRC mice (ICRC is an inbred strain of mouse developed at this Institute) as compared to Balb/c mice, the enhancing activity was not detected in the partially purified, acid soluble intestinal proteins of the ICRC strain. However, studies using polyclonal antibodies against purified EF demonstrated that EF from Balb/c and ICRC intestines are either immunologically identical or closely related to each other although, quantitatively, EF was very low in ICRC mice. RFLP studies indicated that ICRC mice carry a mutation in the coding region of the EF gene resulting in loss of the BamHI. restriction site. On sequencing, a T insertion was found at position 166 from the ATG site thereby causing a disruption in the ORF. This probably results in undetectable levels of enhancing activity. In this paper we report the molecular characterization of the ICRC mouse with respect to theenhancing factor gene     

Abdominal Visceral Fat is Associated with a BclI Restriction Fragment Length Polymorphism at the Glucocorticoid Receptor Gene Locus

Several investigations have suggested that body fat distribution is influenced by nonpathologic variations in the responsiveness to Cortisol. Genetic variations in the glucocorticoid receptor (GRL) could therefore potentially have an impact on the level of abdominal fat. A restriction fragment length polymorphism (RFLP) has previously been detected with the BelI restriction enzyme in the GRL gene identifying two alleles with fragment lengths of 4.5 and 2.3 kb. This study investigates whether abdominal fat areas measured by computerized tomography (CT) are associated with this polymorphism in 152 middle-aged men and women. The less frequent 4.5-kb allele was found to be associated with a higher abdominal visceral fat (A VF) area independently of total body fat mass (4.5/4.5 vs. 2.3/2.3 kb genotype; men: 190.7 ± 30.1 vs. 150.7 ± 33.3 cm², p=0.04; women: 132.7 ± 37.3 vs. 101.3 ± 34.5 cm², p=0.06). However, the association with AVF was seen only in subjects of the lower tertile of the percent body fat level. In these subjects, the polymorphism was found to account for 41% (p=0.003) and 35% (p=0.007), in men and women, respectively, of the total variance in AVF area. The consistent association between the GRL polymorphism detected with BelI and AVF area suggests that this gene or a locus in linkage disequilibrium with the BelI restriction site may contribute to the accumulation of AVF.     

A complete deletion of the factor IX gene and new TaqI variant in a hemophilia B kindred

Summary We report a hemophilia B kindred in which the proband has a complete deletion of the factor IX gene extending a minimum of 80 kilobase pairs (kb) 3 of the gene. This individual has severe factor IX deficiency with no detectable circulating factor IX protein. In common with one previous report, despite a total deletion of the factor IX gene, this patient has not developed antibodies to factor IX. The mother of the proband was found to have a new TaqI variant of the factor IX gene on the nondeletion-bearing X chromosome. The location of the altered TaqI site was found to be 5 of exon IV between residues 9731-9734 and does not affect the function of the factor IX protein. The familial natures of both the variant allele and the deletion were established. In addition a study of this kindred at the DXS99 locus demonstrated the first reported recombination event between this site and the factor IX gene.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

Yeast comparative genetics. A new MEL15 alpha-galactosidase gene of Saccharomyces cerevisiae

To supplement the earlier identified European family of the highly homologous alpha-galactosidase MEL1-MEL11 genes and the African family of the divergent MEL12-MEL14 genes, a new MEL gene (MEL15) was found in several Saccharomyces cerevisiae strains isolated from maize dough in Ghana. Southern blotting and restriction enzyme analysis assigned the MEL15 gene to the African family and mapped it to chromosomes IV/XII, which migrate together in electrophoresis. Tetrad analysis ruled out the MEL15 location in the left arm of chromosome IV or the right arm of chromosome XII, which respectively contain the known MEL5 and MEL10 genes.     

Binding and activation properties of human factor XII, prekallikrein, and derived peptides with acidic lipid vesicles

The binding of human factor XII and prekallikrein to vesicles of various compositions and the relationship to activation of factor XII were studied. Factor XII, factor XIIa, and the 40-kilodalton binding fragment of factor XII bound tightly to all of the negatively charged lipids investigated, including sulfatide, phosphatidylserine, and phosphatidylethanolamine, but not to the neutral lipid phosphatidylcholine. Binding could be reversed by high salt, and the dissociation constant for binding to sulfatide vesicles was in the nanomolar range at an ionic strength of 0.15 M. Prekallikrein did not bind significantly to either sulfatide or phosphatidylethanolamine vesicles under the conditions used. Stopped-flow studies showed that the association rate for the factor XII-sulfatide interaction was biphasic and very rapid; the faster rate corresponded to about 30% collisional efficiency. The kinetics of activation of factor XII was investigated and was in agreement with previous studies; sulfatide promoted activation but phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine did not. Autoactivation rates correlated closely with the packing density of factor XII and factor XIIa on the vesicle surface. In contrast, kallikrein activation of factor XII correlated with the amount of sulfatide-bound factor XII and was relatively insensitive to the density of factor XII on the vesicle surface. When the concentration of factor XII was reduced to only several molecules per vesicle, the autoactivation rate dropped very low whereas kallikrein activation held relatively constant. These results indicated that the autoactivation and the kallikrein activation of factor XII were dependent on different properties of the surface component.     

The Ry-fsto gene from Solanum stoloniferum for extreme resistant to Potato virus Y maps to potato chromosome XII and is diagnosed by PCR marker GP122718 in PVY resistant potato cultivars

A novel locus for extreme resistance to Potato virus Y (PVY), Ry-f_sto, was identified on potato chromosome XII. The gene Ry-f_sto has been introgressed from the wild potato species Solanum stoloniferum. Inheritance of Ry-f_sto in the tetraploid potato population Rysto was consistent with the model of a single, dominant gene. Bulked segregant analysis identified an ISSR (inter-simple sequence repeat) marker UBC 857₉₈₀ linked to Ry-f_sto. This marker mapped to linkage group XII of a reference potato RFLP (restriction fragment length polymorphism) map. Chromosome XII specific RFLP markers were converted into PCR-based STS and CAPS markers and tested for linkage with Ry-f_sto in the population Rysto. CAPS marker GP122₇₁₈ was tightly linked to the resistance gene and was successfully used to identify Polish and German cultivars expressing extreme resistance to PVY. This indicates that the source of Ry-f_sto has been widely utilized in various potato breeding programs and can be monitored by a diagnostic marker in marker-assisted selection.     

Identification of an amino acid essential to the normal assembly of Autographa californica nuclear polyhedrosis virus polyhedra.

We compared the DNA sequence of the Autographa californica nuclear polyhedrosis virus polyhedrin gene with that of the polyhedrin gene from a morphology mutant called M5. A single point mutation was found at the BamHI restriction site within the polyhedrin coding sequence. This point mutation caused a substitution of leucine for proline at amino acid 58 in the M5 polyhedrin. This point mutation was shown to be responsible for both the appearance of cubic polyhedra and the altered mobility of the polypeptide on sodium dodecyl sulfate-polyacrylamide gels by transferring the M5 polyhedrin gene to the wild-type virus by cotransfection. Recombinants were found which assembled cubic polyhedra in infected cells, had the BamHI restriction site missing, and had an altered mobility of their polyhedrin polypeptide. Computed-predicted secondary-structure analysis indicated that the amino acid at position 58 could be critical to the proper folding of polyhedrin.     

A new polymorphic site in the G6PD gene

Summary A polymorphic restriction site has been found in intron 11 of the gene for glucose-6-phosphate dehydrogenase (G6PD). This site is produced by a TC substitution 13 bp upstream of exon 12, producing an NlaIII restriction site. In various populations there was a strong association between a T at nt 1311 of the G6PD cDNA and the presence of the NlaIII restriction site. Among African Americans, however, the presence of a C at nt 1311 was sometimes associated with the presence of a polymorphic NlaIII site.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Apolipoprotein B-100 XbaI gene polymorphism in gallbladder cancer

Genetic polymorphisms in the apolipoprotein B (apoB) gene have been reported to be associated with altered serum lipids and susceptibility to cholesterol gallstones (GS). Gallstones are among the well-known risk factors for carcinoma of the gallbladder (GBC). In the present study, the association between the XbaI polymorphism of the apo B gene was examined in patients with GBC and GS and in normal controls in a north Indian population. DNA samples from patients with GBC (n=153), GS (n=117) and healthy subjects (n=137) were analysed for the apoB-XbaI polymorphism by polymerase chain reaction followed by restriction fragment length polymorphism. The genotype X+/– was less frequent in patients with GBC (39.2%) than in those with GS (68.3%) and in normal subjects (66.4%; P<0.00001). In contrast, there was an increase in the homozygous X–/– genotype in patients with GBC (54.9%) as compared with those with GS (23.9%) and normal subjects (25.5%; P<0.00001). The frequency of the X– allele was found to be significantly increased in GBC patients with or without GS (odds ratio=2.3 and 1.7, respectively). We suggest that the apoB-XbaI gene polymorphism confers susceptibility to carcinoma of the gallbladder under specific environmental conditions.     

Sucrose Synthetase of Rice Grains and Potato Tubers

ABSTRACT

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human βFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.     

Characterization of the Y-Chromosome Sardinian–Corsican Haplotype

The Y chromosome of 523 Italian male subjects was examined for the 49a,f TaqI haplotype XII and for two microsatellites, YCAIIa and YCAIIb. Results were then compared to other populations living in the western Mediterranean basin whom we had previously studied: 419 French (including 328 Corsicans), 46 Italians from Milan, and 73 Tunisians. Haplotype XII is present in 127 out of the 1061 examined samples (11.9%), and most of the haplotype XII subjects are of the compound haplotype YCAIIa-21 and YCAIIb-11. Two peaks of haplotype XII frequencies occur in the north of Sardinia (35%) and in the central area of Corsica (17.4%).     

HLA class I and H ferritin gene polymorphisms in normal subjects and patients with haemochromatosis

Summary The gene for idiopathic haemochromatosis is located on the short arm of chromosome 6 within 1 cM of the HLA-A locus. In this region there are many HLA class I genes, and there may also be a gene for the H subunit of ferritin. Both HLA class I and H ferritin genes are therefore candidates for the abnormal gene in idiopathic haemochromatosis. In 15 unrelated patients the frequency of HLA-A3 was 80% compared with 24% for 600 unrelated individuals from South Wales. The most common haplotype involved is probably HLA-A3, B7. DNA was prepared from leucocytes from 12 of these patients and from 85 normal subjects. After digestion with Taq1, electrophoresis, and Southern blotting, class I sequences were detected by hybridisation to an HLA class I probe (pHLA-A). Of the 34 restriction fragments detected, 22 were polymorphic. Particular fragments correlated with the presence of HLA-A antigens A1, 2, 3, 10, 11, w19, and 28, but there was little correlation with B antigens. Restriction fragment patterns specific for haemochromatosis were not found with TaqI or during less extensive studies with other restriction enzymes. No differences in restriction fragment patterns were found between four patients and four normal subjects apparently homozygous for HLA-A3 and B7. Examination of Southern blotting patterns for genomic DNA from patients and normal subjects with a panel of 12 restriction enzymes and a probe for the H ferritin gene (pDBR-2) revealed no polymorphisms associated with either idiopathic haemochromatosis or particular HLA phenotypes. These studies provide no support for either HLA class I genes or the H ferritin gene as candidates for the haemochromatosis gene.