Genetic polymorphism of apolipoprotein A-IV in the chimpanzee: common deletion of a conserved 12-nucleotide tandem repeat

Apolipoprotein A-IV (apoA-IV protein; APOA4 gene) is structurally polymorphic in various mammalian species, including human, baboon, dog, horse, and mouse. To analyze the extent of genetic variation in the chimpanzee APOA4 gene, we screened 115 common chimpanzees (Pan troglodytes) (86 unrelated wild captured parents and 29 captive-born offspring) using isoelectric focusing followed by immunoblotting for protein polymorphism and using polymerase chain reaction (PCR) assay for DNA polymorphism. At the protein level the unrelated sample of chimpanzees is highly variable, having four alleles, APOA4*1, APOA4*2, APOA4*3, and APOA4*4, with frequencies of 0.192, 0.430, 0.331, and 0.047, respectively. The chimpanzee APOA4 locus, with four common alleles and a gene diversity of 67%, is more variable than previously reported variations in baboons (five alleles with 52% gene diversity) and humans (two alleles with 15% gene diversity). PCR amplification of chimpanzee DNAs, using a pair of human oligonucleotide primers covering a region of 300 nucleotides in the third exon, revealed a common 12-nucleotide deletion (allele frequency = 0.192) that correlates exactly with the APOA4*1 allele detected by isoelectric focusing and immunoblotting. DNA sequencing of the 300-nucleotide PCR amplified product revealed the deletion of 12 nucleotides near the carboxyl terminal region of the mature apoA-IV protein. This in-frame deletion, which codes for and eliminates four amino acids [glutamic acid (GAG), glutamine (CAG), glutamine (CAG), and glutamine (CAG)], occurs in a region that is evolutionarily conserved among rats, mice, chimpanzees, and humans. The partial DNA sequencing of the 3' end of the chimpanzee APOA4 gene revealed 99% identity with the human APOA4 gene.     

Complex beta-satellite repeat structures and the expansion of the zinc finger gene cluster in 19p12

We investigated the organization, architecture, and evolution of the largest cluster ( approximately 4 Mb) of Krüppel-associated box zinc finger (KRAB-ZNF) genes located in cytogenetic band interval 19p12. A highly integrated physical map ( approximately 700 kb) of overlapping cosmid and BAC clones was developed between genetic STS markers D19S454 and D19S269. Using ZNF91 exon-specific probes to interrogate a detailed EcoRI restriction map of the region, ZNF genes were found to be distributed in a head-to-tail fashion throughout the region with an average density of one ZNF duplicon every 150-180 kb of genomic distance. Sequence analysis of 208,967 bp of this region indicated the presence of two putative ZNF genes: one consisting of a novel member of this gene family (ZNF208) expressed ubiquitously in all tissues examined and the other representing a nonprocessed pseudogene (ZNF209), located 450 kb proximal to ZNF208. Large blocks of ( approximately 25-kb) inverted beta-satellite repeats with a remarkably symmetrical higher order repeat structure were found to bracket the functional ZNF gene. Hybridization analysis using the beta-satellite repeat as a probe indicates that beta-satellite interspersion between ZNF gene cassettes is a general property for 1.5 Mb of the ZNF gene cluster in 19p12. Both molecular clock data as well as a retroposon-mapping molecular fossil approach indicate that this ZNF cluster arose early during primate evolution (approximately 50 million years ago). We propose an evolutionary model in which heteromorphic pericentromeric repeat structures such as the beta satellites have been coopted to accommodate rapid expansion of a large gene family over a short period of evolutionary time. [The sequence data described in this paper have been submitted to GenBank under accession nos. AC003973 and AC004004.]     

Instability in the ATCT variable number tandem repeat locus VWF.VNTR I in intron 40 of von Willebrand factor gene

The von Willebrand factor gene intron 40 variable number tandem repeat VWF.VNTR I exhibits 10 alleles making it highly polymorphic and useful for parentage and forensic testing, 45 unrelated families (210 meiotic events) were tested for VWF.VNTR I alleles. One spontaneous mutation was observed in a family member. Haplotype analysis demonstrated that this mutation was due to a gain of one motif repeat by a paternal allele. Sequence analysis confirmed the difference in the number of motif repeats between the proband and the alleles expressed by the parents. This instability emphasizes the importance of demonstrating exclusion in at least two separate loci in parentage testing.     

Repression of the c-myb gene by WT1 protein in T and B cell lines

The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.     

Uninterrupted translation through putative 12-nucleotide coding gap in sequence of carA: business as usual

Previous work of others reported an untranslated stretch of 12 nucleotides in the 5' coding sequence of carA from Pseudomonas aeruginosa. However, N-terminal protein sequencing of carA-lacZ translational fusions shows that these 12 nucleotides are normally translated in a continuous triplet manner, both in P. aeruginosa and in Escherichia coli.     

Band shift analysis of three base-pair repeat alleles in the short tandem repeat locus D12S391

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.     

Major stress protein Hsp70 interacts with NF-kB regulatory complex in human T-lymphoma cells

Utilization of psychiatric in-patient care among 537 new patients was studied in the Department of Psychiatry in Oulu, Finland, during a 3-year follow-up period. Hospitalization during the second and third years of the follow-up was predicted by hospitalization and number of emergency out-patient contacts during the first year of the study, diagnosis of functional psychosis or personality disorder, and previous in-patient care. In total, 5% of the cohort fulfilled our criteria for 'revolving-door' patients. The 'revolving-door' phenomenon was associated with in-patient care at the first contact with the psychiatric services and diagnosis of psychosis or personality disorder. In total, 2% of the cohort became long-stay hospital patients, and this was predicted by psychosis diagnosis. The clinical implications of these findings are that increased attention should be paid to the first assessment of new patients and to the interaction between psychiatric services and patients during the first year of care.     

Transcriptional regulation of vascular endothelial growth factor gene expression in ovarian bovine granulosa cells

From rape (Brassica napus) seedlings proteins able to bind fatty acids and their CoA-esters were purified by gel filtration and cation-exchange chromatography. Among the four proteins detected, one of them (peak IV) appeared purified to homogeneity. This protein is a monomer with a molecular mass of about 9 kDa, as estimated by gel filtration and by polyacrylamide gel electrophoresis. The isoelectric point of the rape protein was higher than 10.5 as determined by chromatofocusing. The pure rape protein appeared furthermore to be able to transfer several phospholipids (phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine) between membranes. The rape protein, having a multifunctional property, was thus called acyl-binding/lipid-transfer protein (AB-LTP). In order to compare this protein to plant lipid-transfer proteins (LTPs), its structure was determined. The amino acid analysis of the rape AB-LTP revealed a high amount of alanine, an absence of histidine and tryptophan and the presence of eight cysteine residues. The N-terminal amino acid sequence of the rape protein revealed a high homology to plant LTPs. These observations led us to propose that the rape AB-LTPs belong to a category of plant proteins interacting with lipids and playing a role in the fatty acid dynamics.     

Inhibition of in vitro proliferation of chronic myelogenous leukemia progenitor cells by c-myb antisense oligodeoxynucleotides

Normal hematopoietic progenitors and acute myelogenous leukemia cells show a differential requirement for the encoded product of c-myb proto-oncogene for proliferation. To determine whether c-myb is also differentially required for the proliferation of hematopoietic progenitors of chronic myelogenous leukemia (CML), mononuclear cells derived from both chronic phase and blast crisis were exposed to c-myb antisense oligodeoxynucleotides and assayed for colony-forming ability. Exposure of CML-BC cells from 12 patients to c-myb antisense oligodeoxynucleotides resulted in significant (p<001) inhibition of leukemia colony formation (average inhibition 63%) and was accompanied by down-regulation of c-myb expression. Colonies derived from CML chronic phase progenitors were virtually unaffected in 10 cases, but down-regulation of c-myb expression was not detected. However, in studies conducted with CD34+ leukemia cells, a subset highly enriched for hematopoietic progenitors, colony formation was inhibited at both disease stages, whereas CFU-GM colony formation derived from normal CD34+ cells was not affected by exposure to c-myb antisense oligodeoxynucleotides. These data suggest that CML chronic phase and blast crisis progenitors are both sensitive to the inhibitory effects of c-myb antisense oligomers, and that the lack of inhibition in partially purified CML-chronic phase progenitors is probably due to inefficient penetration of oligodeoxynucleotides into the clonogenic cells. The preferential effect of c-myb antisense oligodeoxynucleotides on colonies arising from the compartment that includes CML-CD34+ progenitors likely reflects the expansion of a cell population with high proliferative potential and elevated c-myb mRNA levels.     

Directly repeated insertion of 9-nucleotide sequence detected in penicillin-binding protein 2B gene of penicillin-resistant Streptococcus pneumoniae

We investigated the molecular mechanism of 50 penicillin-resistant Streptococcus pneumoniae strains (penicillin: MIC, > or = 0.125 microgram/ml) having neither class A nor class B mutations in the penicillin-binding protein 2B gene (pbp2b). An analysis of the nucleotide sequences of the pbp2b genes from seven strains revealed an unique direct repeat of 9 nucleotides (TGGTATACT) between active-site serine (residue 385) and Ser-X-Asn (residues 442 to 444) motifs. The same insertion was detected in 13 strains.     

Instability of normal (CTG)n alleles in the DM kinase gene

We report on a myotonic dystrophy (DM) family exhibiting instability of normal sized (CTG)n alleles in the DM kinase gene on the non-DM chromosome. At least two mutational events involving normal DM alleles must have occurred in this family; one was characterised as a 34-35 (CTG)n repeat mutation. These findings represent a dissociation between (CTG)n repeat instability and myotonic dystrophy. Furthermore, this family highlights genetic counselling issues relating to the pathogenicity of alleles at the upper end of the normal size range and the risk of further expansion into the disease range.     

4-hydroxynonenal specifically inhibits c-myb but does not affect c-fos expressions in HL-60 cells

4-Hydroxynonenal, an aldehyde produced from lipid peroxidation of cellular membranes, inhibits growth and induces differentiation of HL-60 human leukemic cell line. Since it is highly unstable in the culture medium, its effectiveness is increased when added repeatedly to the cell suspension. We have previously demonstrated that HNE inhibits c-myc but not N-ras expression in HL-60 cells. Here we investigate its effect on the expression of c-myb and c-fos, two early genes involved in the induction of myeloid and monocytic differentiation. Moreover, since c-fos is directly correlated with the intracellular level of cAMP, we also analysed the cAMP concentration after aldehyde treatment. HNE significantly inhibits c-myb expression during and after repeated treatments. A single administration of 1 microM HNE decreases c-myb mRNA at 1 hour whereas 10 microM HNE inhibits c-myb expression from 3 to 6 hours after treatment, and then the expression returns to the control level. By contrast, c-fos expression and intracellular cAMP concentration do not show any significant change after HNE treatments.     

Variability occurs in the inverted repeat region of genomic DNA from bovine herpesvirus 1 respiratory, genital and bovine herpesvirus 5 encephalitic isolates

Restriction fragment length polymorphisms (RFLPs) were detected within BHV1.1, BHV1.2, and BHV5 genomes using the restriction enzyme PstI. The genomic areas of these changes has not been previously reported. Using Southern blot hybridization with DNA probes representing the entire genome of BHV1.1, areas of genomic variation were located for a respiratory isolate (BHV1.1), four vaccine isolates (BHV1.1), a genital isolate (BHV1.2), and two encephalitic isolates (BHV5). The most frequently observed RFLPs of BHV1.1 and BHV1.2 occurred within the internal repeat region and the left terminus of the unique long region. When two separate isolates of the encephalitic BHV5 were compared, RFLPs were detected in the internal and right terminal repeat regions. These are the regions of each genome from which immediate early genes are transcribed. No genomic variation was observed throughout the unique long and unique short regions for all BHV1 and 5 isolates examined.     

CGG repeat interruptions in the FMR1 gene in patients with infantile autism

We determined the CGG repeat length and AGG interruptions in the FMR1 gene in normal Chinese subjects and patients with infantile autism and mild mental retardation. Genomic DNA was investigated by PCR and Southern hybridisation for CGG repeat number and PCR with Mnl I restriction analysis for AGG interruption. Both the normal subjects and the patients with autism have 53 CGG repeats in FMR1, and the majority have two interspersed AGG. Our normal Chinese subjects have a similar number of interspersed AGG as other populations. When compared with the normal subjects, the autism patients have less AGG interruptions and a different pattern of AGG distribution. There was a significant difference in the CGG configurations between normal subjects and patients with autism. The latter had less interspersed AGG, as in fragile X patients, but they did not have fragile X. A study on mentally retarded patients with no infantile autism should also be carried out to ascertain whether mental retardation alone may have contributed to such AGG pattern.     

A CCG repeat polymorphism adjacent to the CAG repeat in the Huntington disease gene: implications for diagnostic accuracy and predictive testing

STUDY HYPOTHESIS: Concentrated aqueous solutions of hydroxocobalamin (OHCob) are given intravenously for the treatment of cyanide poisoning. Because OHCob solutions are intensely red and have peak light absorptions at 352 nm and 525 nm, we investigated whether the presence of OHCob in serum would interfere with various automated, colorimetric chemistry measurements. DESIGN: Selected serum chemistry colorimetric measurements were compared in seven patients, using their own serum as control, with serum containing OHCob at the following concentrations: 100 mg/L, 500 mg/L, and 1,000 mg/L. These concentrations are in the range achieved with therapeutic doses of OHCob when given for cyanide poisoning. MEASUREMENTS AND MAIN RESULTS: Statistically significant alterations in serum values for aspartate aminotransferase, total bilirubin, creatinine, magnesium, and iron were seen in the presence of OHCob. CONCLUSION: The presence of OHCob in serum interferes with several chemistry methodologies, and such interference should be anticipated when this antidote is used.     

Generalized chorea in two patients harboring the Friedreich''s ataxia gene trinucleotide repeat expansion

Recently, a trinucleotide repeat expansion in intron 1 of the frataxin gene on chromosome 9p13 has been identified as the genetic defect in Friedreich's ataxia (FA). We have identified two patients exhibiting generalized chorea in the absence of cerebellar signs who were homozygous for this intron 1 expansion. Chorea as a rare manifestation of FA has previously been controversial. This is the first report of chorea in patients confirmed to have the FA genetic abnormality and broadens further the clinical phenotype associated with the FA genotype.