Heterozygous deletion of ITPR1, but not SUMF1, in spinocerebellar ataxia type 16.

We have previously mapped autosomal dominant spinocerebellar ataxia (SCA) 16 to 3p26, overlapping with the locus of SCA15. Recently, partial deletions of ITPR1 and the neighbouring SUMF1 in the SCA15 and two additional families were reported. In the present study we determined the copy number of these genes by real time quantitative polymerase chain reaction (PCR) and found a heterozygous deletion of exons 1-48 of ITPR1, but not SUMF1 in SCA16. Breakpoint analysis revealed that the size of the deletion is 313,318 bp and the telomeric breakpoint is located in the middle of their intergenic region. Our data provide evidence that haploinsufficiency of ITPR1 alone causes SCA16 and SCA15.

Expression of prokaryotic genes inserted into ColE1 and pVH51 plasmids.

One of the DNA fragments obtained from EcoRI digests of guaA-transducing lambda phage DNA contains the intact bacterial guaA gene at its one end and the lambda phage R gene at the other end. This DNA fragment, named coslambda-guaA, does not contain promoter-operator regions of the gua operon and of the lambda phage R gene, coslambda-guaA DNA fragments were inserted in two different orientations into respective DNAs of EcoRI-cleaved ColE1 and pVH151 (= mini ColE1). Mitomycin C stimulated the guaA enzyme production in the cells harboring the hybrid plasmids with one coslambda-guaA DNA fragment insertion orientation (orientation I), but not the other (orientation II). The lambda-endolysin, product of the lambda phage R gene, was induced by mitomycin C treatment in orientation II, but it was not detected in orientation I. These results suggest that both ColE1 and pVH51 DNAs contain the mitomycin C-responsive promoter which allows transcription of the genes connected with these DNAs.

Expression of a bacterial gene for guanine synthesis inserted into colicin E1 factor by an in vitro recombination.

Regulation of expression of a bacterial guaA gene inserted into colicin E1 DNA by an in vitro recombination was studied under various growth conditions. In Escherichia coli K-12 cells that carried this hybrid ColEl plasmid the level of guaA enzyme activity was not regulated by the concentration of guanine in the medium, but by the number of plasmid DNA copies. The optimal conditions for amplifying the guaA gene product by chloramphenicol treatment were determined. The level of guaA enzyme activity found under the optimal conditions was about 37 times that in extracts of wild-type E. coli cultured in guanine-free medium. The properties of the promoter for the guaA gene and applicability of this hybrid ColEl plasmid for amplification of various gene products were discussed.

Cloning and sequence analysis of a cDNA for the alpha-globin mRNA of carp, Cyprinus carpio.

A cDNA for alpha-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161-170) and its complete nucleotide sequence was determined. The 5' non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an alpha-globin polypeptide consisting of 142 amino acids. The 3' non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp alpha-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.

Identification of five rare mutations including a novel frameshift mutation causing beta zero-thalassemia in Thai patients with beta zero-thalassemia/hemoglobin E disease.

6 out of 14 uncharacterized beta-thalassemia alleles from 187 Thai beta-thalassemia/HbE patients were identified by direct sequencing of DNA amplified by polymerase chain reaction. A novel mutation occurring from an insertion of adenosine in codon 95, which results in a shift of the reading frame with terminator at the new codon 101, was detected in one patient. In addition, two frameshift mutations not previously reported among the Thai population were also detected in 3 patients: one with a deletion of thymidine in codon 15 and two with an insertion of cytidine in codons 27/28. A frameshift mutation that occurred from a cytidine deletion in codon 41 was also found in one patient in this study. The remaining case was an amber mutation, GAG-TAG, in codon 43 in exon 2 of the beta-globin gene. These mutations bring the number of mutations known to be present in the Thai population to a total of 20, 15 of which were detected in beta-thalassemia/HbE patients.

Cloning and sequencing of the cDNA encoding human glutaredoxin.

Glutaredoxin (thioltransferase) is a small, heat-stable protein, which is involved in thiol/disulfide exchange reactions. We have isolated a cDNA that encodes glutaredoxin from a human brain cDNA library. The encoded protein contains 106 amino acids with a calculated molecular mass of 11.76 kDa and an isoelectric point of 8.09. The amino acid sequence deduced from the cDNA is more than 80% identical to those of other mammalian glutaredoxins.

Differential induction of adult and fetal globin gene expression in the human erythremia cell line KMOE.

Induction of globin gene expression in KMOE cells derived from a patient with acute erythremia was studied by Northern blot and S1 analysis. KMOE cells exposed to cytosine arabinofuranoside (Ara-C) synthesized beta-globin gene transcripts, however, in the presence of hemin gamma-globin gene transcripts. An increase in alpha-globin gene transcripts was also detectable in KMOE cells treated with both Ara-C and hemin. Upon exposure to hemin after exposure to Ara-C, or exposure to Ara-C after hemin, there was a 5-10-fold increase in gamma-globin gene transcripts compared to that of cells induced by hemin alone. Neither epsilon nor zeta globin transcripts were detected. The KMOE cell line, therefore, exhibits phenotypic properties of adult and fetal erythroid cells.

A novel CD10-positive erythroid cell line, RM10, established from a patient with chronic myelogenous leukemia.

A novel erythroid cell line, RM10, was established from a long-term bone marrow culture of a patient with chronic myelogenous leukemia (CML). RM10 cells were positive for periodic acid Schiff (PAS), but negative for peroxidase and dual esterase. RM10 cells had la, pre B (CD10), myeloid (CD13, CD14, CD33) and erythroid (glycophorin A) markers, but had no other lymphoid, megakaryocytic, or mesenchymal cell markers. RM10 cells spontaneously synthesized hemoglobin, which was markedly enhanced with hemin. Isoelectric focusing of the cell lysates and northern blot analysis of the total cellular RNA revealed hemoglobin synthesis in the cells. Using 125I-labeled recombinant human erythropoietin (Epo), two classes of Epo receptors were demonstrated in the RM10 cells. However, Epo did affect neither growth nor erythroid differentiation of the cells. RM10 cells rapidly differentiated to monocytic cells in the presence of 12-0-tetradecanoylphorbol-13-acetate, and simultaneously expressed glycoprotein IIb/IIIa. RM10 cells had Philadelphia chromosome (Ph), and expressed p210bcr-abl using immunoprecipitation with anti-c-abl and anti-phosphotyrosine antibodies. These results indicate that the RM10 cells have the characteristics of multipotential hemopoietic cells originating from Ph-positive CML and that high affinity Epo receptor class is not a sufficient condition for Epo responsiveness.

A single amino acid substitution (Met786----Val) in the steroid-binding domain of human androgen receptor leads to complete androgen insensitivity syndrome.

Androgen receptors (ARs) in two Japanese siblings with complete androgen insensitivity syndrome were characterized, and their molecular bases were investigated. Androgen binding was undetectable in cultured pubic skin fibroblasts from the patients by whole cell assay. Sequence analysis of exons B-H, which encode the DNA- and steroid-binding domains, of the AR gene from these patients using polymerase chain reaction revealed a single nucleotide substitution in exon F, resulting in an amino acid change at 786 from methionine (ATG) to valine (GTG) within the steroid-binding domain of AR. Reconstruction of this mutation by site-directed mutagenesis into human AR cDNA followed by expression in COS-1 cells led to production of the same amount and the same molecular mass of immunodetectable AR protein as those found with expression of the normal human AR cDNA. However, in contrast to wild-type AR expressed in COS-1 cells, the mutant AR showed markedly low affinity of androgen binding by whole cell assay. These results suggest that androgen resistance in these patients is due to the point mutation in the steroid-binding domain of the AR.

Mode of activation of the GC box/Sp1-dependent promoter of the human NADH-cytochrome b5 reductase-encoding gene.

Many eukaryote promoters, particularly those for so-called housekeeping genes, have multiple GC boxes which are the binding sites of the transcription factor, Sp1. It has been proposed that Sp1 binds to the multiple GC boxes, and then the GC box-bound Sp1 interact with each other to synergistically stimulate transcription. Here, we describe a Sp1-dependent promoter which does not necessarily fit the synergistic activation mechanism. The promoter of the human NADH-cytochrome b5 reductase-encoding gene (CYTB5R) possesses five potential GC box sequences. Deletion and mutagenesis studies coupled with CAT assays revealed that three out of five GC box-like sequences were functionally active and activated transcription additively (rather than synergistically). Our results suggested that Sp1-mediated activation of transcription occurs in a promoter context-dependent manner.

The organization and the complete nucleotide sequence of the human NADH-cytochrome b5 reductase gene.

The organization and the complete nucleotide (nt) sequence of the b5R gene encoding human NADH-cytochrome b5 reductase (b5R; EC 1.6.2.2) have been determined by a combination of restriction mapping and nt sequence analysis of overlapping genomic DNA clones. The entire gene is about 31 kb in length and contains nine exons and eight introns. Exon 2 contains the junction of the membrane-binding domain and the catalytic domain of b5R, indicating that two forms of b5R, a soluble and a membrane-bound form, are generated by post-translational processing. The 5' portion of the b5R gene lacks the canonical 5' transcriptional regulatory elements, but contains five copies of the GC box sequence G-G-G-C-G-G. While the average G + C content of the b5R gene is 55%, that of the 5' portion of the gene is extraordinarily high (86%). The CpG dinucleotide sequence was found at a very high frequency in this G + C-rich region. These structural features are very similar to those of the regulatory regions of constitutively expressed 'housekeeping' genes. Several transcription start points were identified by the primer extension experiment. Seventeen complete and twelve incomplete Alu family sequences were found in introns. An uncanonical polyadenylation signal was detected in the 3'-untranslated region of the gene as A-G-T-A-A-A instead of A-A-T-A-A-A.

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake.

Inhibitors (PLIs) against snake venom gland phospholipases A2 (PLA2s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA2 isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.

Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes.

Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A2 (PLA2) isozymeencoding genes, gPLA2-o1, gPLA2-o2 and gPLA2-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA2-o2 and gPLA2-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA2 isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA2 isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA2 isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA2 isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA2 isozyme genes appears to have been brought about by natural selection for point mutations.

Structures of genes encoding TATA box-binding proteins from Trimeresurus gramineus and T. flavoviridis snakes.

A cDNA encoding the Trimeresurus gramineus (Tg; green habu snake) TATA-box-binding protein (TgTBP) was cloned and sequenced. The cDNA encodes a 33-kDa protein with an extensive sequence similarity to those derived from other organisms, except for the N-terminal domain. Genes encoding TgTBP and Trimeresurus flavoviridis (Tf; habu snake) TBP (TfTBP) were isolated using a TgTBP cDNA and their nt sequences were determined. They are the first TBP genes entirely sequenced in higher animals. Both genes span over 15 kb and are constructed from eight exons and seven introns. Comparison of the loci of introns on the aligned amino-acid sequences of TBP from six organisms (Tg, Tf, mouse, Arabidopsis thaliana, Schizosaccharomyces pombe and Acanthamoeba castellanii) indicated that there are three highly conserved loci in the C-terminal domain.

Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A2 isozymes.

Five inhibitors (PLI-I-V) against Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) venom phospholipase A2 (PLA2) isozymes have been isolated from its serum. PLI-I, which is composed of two repeated three-finger motifs, and PLI-IV and PLI-V, which contain a sequence similar to the carbohydrate recognition domain (CRD) of C-type lectins, were expressed in the forms fused with glutathione S-transferase (GST). The resulting GST-PLIs showed ability to bind to three Tf venom PLA2 isozymes. The binding study with the truncated forms indicated that one of two three-finger motifs of PLI-I was able to bind to PLA2 isozymes. The N-terminal 37-amino acid fragment and the CRD-like domain of PLI-IV and PLI-V were bound to PLA2 isozymes. On the other hand, their C-terminal 12-amino acid segment also associated with PLA2 isozymes. When either of two units of a hydrophobic tripeptide in this sequence was replaced by trialanine, the binding was completely abolished, indicating that the C-terminal hydrophobic cores of PLI-IV and PLI-V were critically responsible for the binding to venom PLA2 isozymes.

Accelerated evolution of crotalinae snake venom gland serine proteases.

Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein-coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

Significance of pH on differentiation of human erythroid cell lines.

As physiological factors and compounds, BSA and/or higher pH of the culture medium could induce the erythroid differentiation of cells. Optimum pH values of the culture medium for higher spontaneous differentiation of KU-812 and K562 cells after 7 days cultivation were 7.5 and 7.6, respectively. The synergistic effects on the differentiation were observed by exposure to hemin under the higher pH condition. In the presence of BSA, 25% of KU-812 and 40% of K562 cells became benzidine positive. Synergistic effects of BSA and the higher pH of the medium were also observed.

Hematopoietic growth factors (BPA and Epo) induce the expressions of c-myc and c-fos proto-oncogenes in normal human erythroid progenitors.

We investigated serial expressions of eight proto-oncogenes during in-vitro differentiation of normal human burst-forming unit, erythroid (BFU-E), and found that c-myc and c-fos are expressed in progenies of BFU-E. The expressions of the two proto-oncogenes correlated to the replating efficiency and adversely to erythroid differentiation. The absence of hematopoietic growth factors decreased the expressions, but the addition of erythropoietin together with burst promoting activity induced a re-expression of the c-myc and c-fos after 2 h of incubation. These observations suggest that the c-myc and c-fos proto-oncogenes have a physiological role in the proliferation of erythroid progenitors and that activations of the two proto-oncogenes are early cellular events after the stimulation by hematopoietic growth factors.

Significance of alpha-1-antitrypsin mRNA expression in esophageal carcinoma.

Alpha-1-antitrypsin (alpha 1AT) is present not only in the normal gastrointestine, but in malignant gastrointestinal tissue as well. We studied the expression of alpha 1AT mRNA in 30 cases of esophageal carcinoma using a Northern blot analysis. In addition, we also examined the expression of matrix metalloproteinase 7 (MMP7) mRNA in the latest 15 cases by reverse transcriptase-polymerase chain reaction (RT-PCR) method to clarify the relationship between the alpha 1AT and MMP7. In 25 of the 30, the expression of alpha 1AT mRNA in esophageal carcinomatous tissue (T) was lower than that in the corresponding normal tissue (N) of the esophagus. The T/N ratio of alpha 1AT mRNA expression showed a significant inverse correlation with the depth of tumor invasion, lymph node metastasis and stage of disease (p=0.042, p=0.0001 and p=0.002, respectively). The T/N ratio of alpha 1AT bad a converse correlation with that of MMP7 (p=0.034). The current study thus suggested that i) the lower expression of T/N expression may be a marker for the biological aggressiveness of esophageal carcinoma, and ii) the reduction of alpha 1AT may be partially correlated with the overexpression of MMP7 in tumor tissue.

Differential decreases in c-fos and aldolase C mRNA expression in the rat cerebellum after repeated administration of methamphetamine.

The effects of repeated methamphetamine administration on c-fos mRNA and aldolase C (Zebrin) mRNA expression in the rat cerebellum were investigated. A single dose of methamphetamine induced c-fos mRNA expression in granule and Purkinje cells of both anterior and posterior lobes. In the posterior lobe, in particular, c-fos mRNA signals were distributed in a parasagittal organization, like Zebrin bands. Repeated methamphetamine injections reduced methamphetamine-induced c-fos mRNA signals in the anterior hemisphere and in part of the posterior vermis (lobule VII) and posterior hemisphere. Aldolase C mRNA signals in Purkinje cells decreased only in lobules where methamphetamine-induced c-fos signals were not reduced (lobules VI and IX). Therefore, differential decreases in c-fos mRNA and aldolase C mRNA expression after repeated methamphetamine administration depend upon the localization of Purkinje cells in the cerebellum. Since c-fos mRNA and aldolase C mRNA expressions are markers of excitability and the metabolic state of Purkinje cells, respectively, hypofunction of inhibitory Purkinje cells could be induced if methamphetamine is repeatedly injected. Since repeated methamphetamine administration in this experimental paradigm increased horizontal movement and the rearing activity of rats, the hemisphere of the cerebellum may be involved in development of methamphetamine-induced motor behavioral sensitization in addition to the striatum and the nucleus accumbens.