The variable number of tandem repeats in the renin gene of rats and possible significance in hypertension

The variable number of tandem repeats (VNTR) in the first intron of renin gene for the spontaneously hypertensive rat (SHR), its controls Wistar-Kyoto (WKY), renal hypertensive rat, and Sprague-Dawley rat (SD) were compared by polymerase chain reaction (PCR) method. An analysis of VNTR from WKY, Wistar and SD showed that there are two different renin gene alleles and three genetypes 2.0kb/2.0kb, 2.0kb/1.8kb, 1.8kb/1.8kb. The genetype from renal hypertensive rats is same as those seen in the normal controls. However, compared with the WKY, Wistar and SD genes, a "deletion" of approximately 1.0kb was found in the first intron of the SHR renin gene. Our results strongly suggest that the cause and mechanism of elevated blood pressure is complex, and the molecular basis of the genetic-prone hypertension is existed.     

Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (> 8 bp) deletions or additions.     

The Cystatin-C gene is not linked to early onset familial Alzheimer's disease

The APP717 mutations discovered in only a few early onset Alzheimer's disease (AD) families have confirmed the genetic heterogeneity of this disorder. To identify the other gene(s) involved in the disease we selected the protease inhibitor, Cystatin-C, as a candidate gene. Cystatin-C is an amyloidogenic protein causing hereditary cerebral haemorrhage with amyloidosis-Icelandic type (HCHWA-I). It is localised with the beta-amyloid peptide in the arterial walls of AD brains. We have analysed the segregation of a polymorphic marker in this gene in 8 early onset AD families. Two early onset families showed clear non-segregation of the marker with the disease. When the 8 families are analysed together (assuming only one other gene is involved), they present exclusion linkage criteria. These data indicate that Cystatin-C is not the site of the defect in 2 families and is not likely to be in the other families analysed. We conclude that the deposition of Cystatin-C in AD is a secondary event in the disease process, and that this gene is not pathogenic in familial AD.     

nec1, a gene conferring a necrogenic phenotype, is conserved in plant-pathogenic Streptomyces spp. and linked to a transposase pseudogene

We are investigating the genetic basis for, and evolution of, plant pathogenicity in Streptomyces spp. The plant-pathogenic species S. scabies, S. acidiscabies, and S. turgidiscabies cause the scab disease of potato and produce the phytotoxins, thaxtomins. Forty-three Streptomyces strains representing the three species were evaluated; all thaxtomin A-producing Streptomyces strains were pathogenic on potato tubers and all but one hybridized to nec1 and ORFtnp, two genes previously cloned from S. scabies ATCC 41973. nec1 confers a pathogenic phenotype on S. lividans TK24, a nonpathogen, and ORFtnp is a transposase pseudogene located 5' to nec1. The eight nonpathogenic strains tested neither produced thaxtomin A nor hybridized to nec1. ORFtnp and nec1 occurred on a single PvuII restriction fragment in all thaxtomin A-producing Streptomyces strains. The nucleotide sequences of the homologs of nec1 and ORFtnp from two pathogenic strains each of S. scabies, S. acidiscabies, and S. turgidiscabies were identical; oligonucleotide primers specific to this gene amplified homologs from all strains that hybridized to nec1. We propose that nec1 and ORFtnp have been horizontally mobilized from S. scabies to S. acidiscabies and S. turgidiscabies, and that nec1 is involved in pathogenicity and physically linked to the thaxtomin A biosynthetic genes.     

Part of the RBM gene cluster is located distally to the DAZ gene cluster in human Yq11.23

Normal human Y and inverted Y chromosomes were chosen for physical fluorescence in situ hybridization (FISH) mapping of RBM and DAZ probes for the relative positioning of the RBM and DAZ gene clusters in interval 6 of the human Y chromosome. The inversion breakpoint in Yq11.23 turned out to be distal to the DAZ gene cluster, as the entire DAZ signal appears in the short arm of the inv(Y) chromosome. On the contrary, this inversion breakpoint in Yq11.23 divides the RBM signal cluster, leaving a weaker signal on the long arm while bringing the main RBM signal to the short arm of the inv(Y) chromosome. Thus, it can be concluded that, in contrast to previous claims, part of the RBM gene cluster is located distally to the DAZ gene cluster in deletion interval 6 of the human Y chromosome.     

The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster

Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.     

Oligoribonuclease is encoded by a highly conserved gene in the 3'-5' exonuclease superfamily

Oligoribonuclease, a 3'-to-5' exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts of Escherichia coli. The purified protein is an alpha2 dimer of 40 kDa. NH2-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as yjeR (o204a), a previously reported open reading frame located at 94 min on the E. coli chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of yjeR led to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose that yjeR be renamed orn. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.     

A novel mechanism of in vivo ras gene activation involving tandem duplication of coding sequences

A series of weakly transforming c-K-ras genes have been detected in spontaneously occurring and chemically induced mouse adenomas. DNA sequence analysis of these weakly transforming ras oncogenes showed that activation occurred by a novel mechanism involving duplication of nine or ten codon segments flanking codon 61 in exon 2. The codon repetitions in exon 2 are directly preceded by a number of potentially recombinogenic DNA sequences which may have been involved in the genesis of the codon repetitions through mechanisms involving recombination or DNA slippage. Duplication of DNA sequences such as those observed in the mouse c-K-ras gene may represent a new mechanism for both tumor suppressor gene inactivation and proto-oncogene activation.     

The GABAA receptor alpha 6 subunit gene (Gabra6) is tightly linked to the alpha 1-gamma 2 subunit cluster on mouse chromosome 11

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by hyperplasia and neoplasia in several endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a 900-kb region on chromosome 11q13. The human phosphatidylinositol-specific phospholipase C beta 3 (PLC beta 3) gene, which is located within this region, was considered to be a good candidate for the MEN 1 gene. In this study, the structure and expression of the PLC beta 3 gene in MEN 1 patients were investigated in more detail, to determine its potential role in MEN 1 tumorigenesis. Southern blot analysis, using blood and tumor DNA from affected persons from seven different MEN 1 families, did not reveal structural abnormalities in the PLC beta 3 gene. To detect possible point mutations, or other small structural aberrations, direct sequencing of PLC beta 3 cDNAs from two affected persons from two different MEN 1 families was performed, but no MEN 1-specific abnormalities were revealed. Several common nucleotide sequence polymorphisms were detected in these cDNAs, proving that both alleles of the PLC beta 3 gene were expressed and analyzed. In conclusion, these results exclude the PLC beta 3 gene as a candidate gene for MEN 1.     

The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression

We have isolated and determined DNA sequence for the 5'-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5'-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5'-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5'-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli beta-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5' intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.     

The human homologue for the Caenorhabditis elegans cul-4 gene is amplified and overexpressed in primary breast cancers

Amplification is a key mechanism whereby a cancer cell increases the message level of genes that confer a selective advantage when they are overexpressed. In breast cancer, there are many chromosome regions present in multiple copies relative to overall DNA copy number (amplicons), and their target genes are unknown. Using differential display, we have cloned and sequenced the full coding region of a candidate amplicon target gene located on chromosome 13. This candidate is the human homologue of the Caenorhabditis elegans cul-4 gene, cul-4A, a member of the novel cullin gene family, which is involved in cell cycle control of C. elegans. cul-4A was amplified and overexpressed in 3 of 14 breast cancer cell lines analyzed, and it was overexpressed in 8 additional cell lines in which it was not amplified. The latter observation, indicating that its overexpression can occur by mechanisms other than gene amplification, suggests that cul-4A plays a key role in carcinogenesis. Moreover, cul-4A was found to be amplified in 17 of 105 (16%) cases of untreated primary breast cancers, and 14 of 30 cases analyzed (47%) were shown by RNA in situ hybridization to overexpress cul-4A. These results suggest that up-regulation of cul-4A may play an important role in tumor progression.     

The use of amplified variable number of tandem repeats (VNTR) in the detection of chimerism following bone marrow transplantation. A comparison with restriction fragment length polymorphism (RFLP) by Southern blotting

A 49-year-old woman patient with atypical myelodysplastic syndrome (MDS) showing a der(3)t(3;12)(q21;p13), and der(12)t(3;12)(q21;p13)inv(3)(q21q26) as an acquired chromosomal abnormality in the bone marrow is described. The chromosomal breakpoints of the presented complex aberration with combination of the inv(3)(q21q26) and t(3;12)(q21;p13) were defined by fluorescence in situ hybridization (FISH) with yeast artificial chromosomes (YACs). The inv(3) is a relatively frequent chromosomal rearrangement in patients with myeloid malignancies and dysmegakaryopoiesis and t(3;12)(q26;p13) has also been reported as a recurrent abnormality in MDS and in blast crisis of chronic myelogenous leukemia (CML). Whereas the t(3;12), inv(3), and t(3;3) are associated with a very poor prognosis, our patient surprisingly had a mild clinical course.     

The lunatic fringe gene is a target of the molecular clock linked to somite segmentation in avian embryos

The most obvious segments of the vertebrate embryo are the trunk mesodermal somites which give rise to the segmented vertebral column and the skeletal muscles of the body. Mechanistic insights into vertebrate somitogenesis have recently been gained from observations of rhythmic expression of the avian hairy-related gene (c-hairy1) in chick presomitic mesoderm (PSM), suggesting the existence of a molecular clock linked to somite segmentation ([1]; reviewed in [2]). Here, we show that lunatic Fringe (IFng), a vertebrate homolog of the Drosophila Fringe gene, is also expressed rhythmically in PSM. The PSM expression of IFng was observed as coordinated pulses of mRNA resembling the expression of c-hairy1. We show that c-hairy1 and IFng expression in the PSM are coincident, indicating that both genes are responding to the same segmentation clock. The genes were found to differ in their regulation, however; in contrast to c-hairy1, IFng mRNA oscillations required continued protein synthesis, suggesting that IFng could be acting downstream of c-hairy1 in the clock mechanism. In Drosophila, Fringe has been shown to play a role in modulating Notch-Delta signalling [3,4], a pathway which in vertebrates has been implicated in defining somite boundaries [5-9]. These observations place the segmentation clock upstream of the Notch-Delta pathway during vertebrate somitogenesis.     

The blood contribution to early myocardial reperfusion injury is amplified in diabetes

Cardiovascular disease is excessive in diabetes, and blood cell function is altered. It is not clear, however, if alterations in the blood contribute to the excessive cardiovascular complications of this disease. In this study, we compared the contribution of nondiabetic and diabetic blood to myocardial reperfusion injury. The recovery of cardiac contractile function following no-flow ischemia was studied in isolated diabetic and nondiabetic rat hearts perfused with diabetic or nondiabetic diluted whole blood. Hearts were isolated from 10- to 12-week-old diabetic (streptozotocin, 65 mg/kg, i.v.) and nondiabetic rats and perfused with a Krebs-albumin-red cell solution (K2RBC, Hct 20%). After a 30-min pre-ischemic control period, during which cardiac pump function was evaluated, diabetic and nondiabetic hearts were perfused for 5 min with diluted whole blood (DWB; Hct 20%) collected from either diabetic or nondiabetic donor animals. Coronary flow was then stopped and the hearts subjected to 30 min of no-flow ischemia. Following ischemia, the hearts were reperfused with the K2RBC perfusate. Cardiac contractile function was evaluated throughout the 60-min reperfusion period. Six groups were studied: diabetic and nondiabetic hearts perfused before ischemia with either K2RBC, nondiabetic DWB (NDDWB), or diabetic DWB (DDWB). Perfusion with DWB prior to ischemia impaired the recovery of contractile function in all cases. The impairment to recovery was greater with DDWB than with NDDWB. Although diabetic hearts perfused with K2RBC throughout recovered quite well, the effect of DDWB perfusion in the diabetic hearts was dramatic. In an effort to determine why diabetic blood impaired functional recovery, measures of blood filterability and the generation of reactive oxygen species (ROS) were made. We found that diabetic blood was less filterable than nondiabetic blood; that is, the diabetic blood cells tended to plug the 5-microm filter pores more readily than the nondiabetic blood cells. Also, we found that the diabetic blood was capable of generating significantly greater ROS (oxygen free radicals) than nondiabetic blood (P < 0.05). These findings suggest that the blood contribution to myocardial reperfusion injury is amplified in diabetes. A tendency for diabetic blood cells to plug capillary-sized pores and show enhanced oxygen free radical production may account for the excessive contribution of diabetic blood to reperfusion injury in the heart.