Genomic Organization and Mutation Analysis ofp73in Oligodendrogliomas with Chromosome 1 p-Arm Deletions

p73, a protein having substantial structural and functional similarity to p53, has recently been identified and demonstrated to be a potential tumor suppressor. Its location on human chromosome 1p36.33 implicates p73 as a candidate for neuroblastoma. Like neuroblastoma, oligodendrogliomas also show a high frequency of deletions in chromosome 1p36.3. To determine whetherp73is a potential tumor suppressor gene involved in the development of oligodendrogliomas, we performed mutation analysis ofp73in oligodendrogliomas with chromosome 1 p-arm deletions. We first determined the genomic organization and the intron–exon boundary sequences of thep73gene by long PCR, vectorette PCR, and Southern hybridization. This gene spans about 65 kb with a large first intron. Primer pairs for the amplification of each of the 13 p73 encoding exons were designed in corresponding introns. The amplicons were then analyzed using the denaturing high-performance liquid chromatography system for mutations in thep73gene. Twenty oligodendroglioma samples with 1p36.3 deletions were screened, but no mutations were detected except for several polymorphisms. It is thus clear thatp73is not a candidate gene for oligodendroglioma despite its location in the frequently deleted 1p36.3 region.     

Investigations on the PGM (phosphoglucomutase) polymorphism by isoelectric focusing inDrosophila melanogaster

Pgm allele frequencies of 383 individuals were determined in a sample ofDrosophila melanogaster from three laboratory Sardinian populations, using the techniques of standard electrophoresis, heat denaturation, and isoelectric focusing. The analysis of the progeny obtained from informative crosses showed that the isoelectric focusing patterns segregate in a Mendelian way. ThePgm ^1.00 andPgm ^0.70 electrophoretic alleles displayed different isoelectric points, whereas thePgm ^1.00,tr andPgm ^1.00,ts isoelectrophoretic alleles could not be differentiated when tested by isoelectric focusing. Moreover, thePgm ^0.70,ts allele was split into two classes, with isoelectric points ofpH 6.4 andpH 6.6.     

Vitamin B12 transport in Escherichia coli K12 does not require the btuE gene of the btuCED operon

Summary Transport of vitamin B₁₂ across the cytoplamic membrane ofEscherichia coli requires the products ofbtuC andbtuD, two genes in thebtuCED operon. The role ofbtuE, the central gene of this operon, was examined. Deletions withinbtuE were constructed by removal of internal restriction fragments and were crossed onto the chromosome by allelic replacement. In-frame deletions that removed 20% or 82% of thebtuE coding region did not affect expression of the distalbtuD gene. These nonpolar deletions had little effect on vitamin B₁₂ binding (whole cells or periplasmic fraction) and transport. They did not affect the utilization of vitamin B₁₂ or other cobalamins for methionine biosynthesis, even in strains with decreased outer membrane transport of vitamin B₁₂. ThebtuE mutations did not impair adenosyl-cobalamin dependent catabolism of ethanolamine or repression ofbtuB expression. Thus, despite its genetic location in the transport operon, thebtuE product plays no essential role in vitamin B₁₂ transport.     

Deletion of theAlu-VpA/MycL1 (1p34.3) locus is a negative prognostic sign in human colorectal cancer

We examined deletions of the short arm of chromosome 1 and aberrations of the microsatellite locusAlu-VpA/MycL1 (1p34.3) in human primary colorectal adenocarcinomas. Cytogenetically discernible deletions in 1p were found in 45% (14/31) of informative tumors. The 1p-tumors commonly exhibited a polyploid karyotype (FisherP ₁=0.023) and a larger number of rearranged chromosomes (P ₂=0.045) versus those without 1p deletions. The 1p deletions often combined with chromosome 5 monosomy (χ²=6.24; p=0.013), chromosome 15 monosomy (χ²=4.20;p=0.040), and 11q deletions (P ₂=0.035). Among the 50 carcinomas, 11 (22%) showedAlu-VpA/MycL1 instability, and 14% (6/43 informative) had lost theAlu-VpA/MycL1 allele. The genetic alterations thus revealed were collated with the clinical and morphological features of the tumors. The loss of the 1p material was shown to be correlated with marked karyotype aberrations in colorectal tumors, andAlu-VpA/MycL1 allele deletions were tightly associated with relapses or metastasis within 30 months after surgery.     

Detection of 14 alleles derived from the MHC class I<Emphasis Type="Italic"> A</Emphasis> locus in cynomolgus monkeys

A basic understanding of the major histocompatibility complex (MHC) class I, which, together with T-cell receptors, is a key player in antigen recognition by cytotoxic T lymphocytes, is necessary to study the cellular immune response to intracellular pathogens. The MHC has hardly been reported in cynomolgus monkeys (Macaca facicularis), although cynomolgus monkeys have been frequently used as the surrogate animal model. We attempted to determine the nucleotide sequences of the MHC class I A locus of cynomolgus monkeys (Mafa-A) and eventually 34 independent sequences of Mafa-A were obtained from 29 cynomolgus monkeys. These 34 sequences were classified into 14 Mafa-A alleles according to the results of phylogenetic analyses using the neighbor-joining method. One to three Mafa-A alleles were obtained from a single animal. We also tried to establish a multiplex PCR-SSP method for convenient typing of Mafa-A alleles. cDNA from a family of cynomolgus monkeys, which is composed of four sirs and four dams, were examined by multiplex PCR-SSP. The result of multiplex PCR-SSP showed that an individual cynomolgus monkey had two or three Mafa-A alleles, suggesting that the A locus of cynomolgus monkeys might be duplicated.     

Efficient transformation of the medicinal mushroomGanoderma lucidum

Ganoderma lucidum is a well-known and important medicinal mushroom, but its genetic modification has not been reported. We developed an efficient procedure for isolation and regeneration of protoplasts fromG. lucidum. To construct a vector for high-level expression of heterologous genes inG. lucidum, the 1.4-kb regulatory region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) was isolated from the genomic DNA ofLentinus edodes, and theGPD promoter was fused to the β-glucuronidase (GUS) and bialaphos resistance (bar) genes. Using the resulting construct, p301-bG1, an efficient transformation system based on electroporation was established forG. lucidum. GUS expression was observed among transformants conferring bialaphos resistance, indicating that theL. edodes GPD promoter can be used for expression of exogenous genes inG. lucidum. We also studied green fluorescent protein (GFP) as another reporter for transformation ofG. lucidum. TheL. edodes GPD promoter was fused respectively to theGFP andbar genes, and the resulting construct, p301-bg, was introduced intoG. lucidum. StableGFP expression in transformants was detectable by fluorescence microscopy, suggesting the suitability ofGFP as a reporter system in transformation of this mushroom. This is the first report of an efficient transformation system forG. lucidum using different reporters, paving the way for genetic modification of this famous medicinal mushroom.     

Regulation of the expression of thePseudomonas stutzeri recA gene

With the aid ofrecA-lacZ fusion strains, thein vivo regulation of thePseudomonas stutzeri recA gene has been studied. It is shown that expression of this gene can be induced with a variety of DNA damaging agents, as well as with agents that interfere with DNA replication. For this induction, the presence of an active RecA protein is essential. Sequence analysis of the promoter region of theP. stutzeri recA gene showed that its open reading frame is preceded by an SOS-box, suggesting a regulation of its expression, similar to the regulation ofrecA expression inEscherichia coli.Abbreviation MMS Methyl-methane-sulphonate     

Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

Deletions of the YNZ22and Alu-VpA/MycL1Loci and Post-Surgery Prognosis in Human Colorectal Adenocarcinoma

Since deletions of the short arm of chromosome 17 are the most common genetic defects in human colorectal carcinoma (CC), we tested the YNZ22locus (D17S30, 17p13.3) for loss of heterozygosity (LH) in adenocarcinoma and in the normal colonic mucosa of 49 CC patients, and studied the association of LH with clinicomorphological features of the tumor. Allele frequency distribution of YNZ22did not differ for the patients and healthy people. LH in YNZ22in the tumor was found in 33% (13/39) of all informative cases, its frequency being thrice higher in men than in women (²= 5.21, p= 0.022). The defect was associated with moderate or poor histological differentiation (P ₂= 0.0055) and polyploidy >3n(P ₂= 0.0035) of tumor cells and with high incidence of post-surgery relapse or metastasis. Analysis of both YNZ22and Alu-VpA/MycL1(1p34.3) loci in the tumor allowed reliable relapse prognosis in 76% of the CC patients. The probability of post-surgery relapse or metastasis was estimated at no less than 67% for patients with LH in at least one of the two loci in the tumor, and at somewhat more than 20% for patients without LH.     

Interactions of thePolycomb group of genes with homeotic loci ofDrosophila

Summary Members of thePolycomb (Pc) group of genes are required for the correct determination of segment identity, and are thought to be negative regulators of thebithorax andAntennapedia complexes. This hypothesis has been tested molecularly for only some members of thePc group. Here, we examine the distribution ofUltrabithorax (Ubx),Antennapedia (Antp), andSex combs reduced (Scr) proteins in the epidermis, central nervous system, and midgut of embryos homozygous for mutations in tenPc group genes. We show that zygotic loss of mostPc group genes causes ectopic expression ofUbx andAntp, but that there are differences in time and tissue-specificity. FivePc group mutations lack midgut constrictions and also exhibit ectopic or suppressedUbx expression and suppression ofAntp expression. Distribution ofAntp is upset earlier than distribution ofUbx in the central nervous system of everyPc group mutant affecting both genes. Loss of the zygotic products ofPolycomb, extra sex combs, andAdditional sex combs cause ectopic expression ofScr in epidermis, and allPc group genes exceptPsc have suppressedScr expression in the nervous system. These results are discussed with respect to the function of thePc group.     

Conjugative plasmid pIP501 undergoes specific deletions after transfer from<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> to<Emphasis Type="Italic"> Oenococcus oeni</Emphasis>

Conjugal transfer of plasmids pIP501 and its derivative pVA797 from Lactococcus lactis to Oenococcus oeni was assayed by filter mating. Plasmid pIP501 was transferred to a number of O. oeni strains whereas a single transconjugant of O. oeni M42 was recovered when pVA797 was used. Physical analysis of the transconjugant plasmids revealed that pIP501 and pVA797 underwent extensive deletions in O. oeni that affected the tra region (conjugal transfer) and SegB region (stability). All derivatives showed segregational instability in O. oeni, but were stably maintained in L. lactis. These differences correlated with the different plasmid copy numbers and the extent of deletions within the SegB region.Abbreviations CAT Chloramphenicol acetyltransferase - MLS Macrolides-lincosamides-streptogramin B resistance     

Larval mortality of pine sawflies [Hymenoptera: Diprionidae] in relation to pollution level: A field experiment

Larval mortality ofNeodiprion sertifer (Geoffroy),Diprion pini (L.) andGilpinia pallida (Klug) were studied in field experiments around a factory complex in southwestern Finland. Larval colonies were transferred on the shoots of Scots pines growing at different distances from the emission source. Larval mortality was highest near the factories. InN. sertifer, larval mortality caused by the nuclear polyhedrosis virus was higher and cocoon mortality caused by parasitoids was lower near the pollutant source. The most abundant parasitoid species wereSynomelix scutulata (Hartig) andLamachus eques (Hartig). 16–67% of theN. sertifer, 0–5% of theD. pini and the 73–100% ofG. pallida cocoons contained parasitoids oviposited during the larval period of the sawflies.      

Complete sequence of p53 gene in 20 patients with lung cancer: comparison with chemosensitivity and immunohistochemistry

In this study the entirep53 complementary DNA has been sequenced in 20 non-small cell lung carcinomas (NSCLC) and the results correlated with chemosensitivity, immunohistochemistry and clinical data. Ten patients had mutations inp53, 8 missense mutations and 2 nonsense mutations. The method discovered two mutations never described previously and two other mutations that have never been described before in connection with NSCLC tumours. Chemosensitivity data, according to a short-term assay (FMCA), indicated that tumours with p53 mutation were more resistant to cisplatin and cyclophosphamide. Immunohistochemical studied demonstrated a 70% concordance between over-expression of p53 protein and mutation inp53. No conclusions or trends could be drawn from the immunohistochemical studies ofBcl-2 andBax.     

Decreased production ofpara-hydroxypenicillin V in penicillin V fermentations

Summary Penicillin V (phenoxymethyl penicillin) is produced by industrial strains ofPenicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain ofP. chrysogenum produces an undesirable penicillin byproduct,para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, viapara-hydroxylation of POAc and subsequent incorporation of thep-OH phenoxyacetic acid into the penicillin molecule. Most of thep-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level ofp-OH penicillin V produced by the control strain ranges up to 10–15% of the total penicillins produced. 3-Phenoxypropionic acid andp-bromophenylacetic acid partially inhibit the formation ofp-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels ofp-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which producep-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.     

Contribution of genetic distances studies to the taxonomy ofAteles,particularlyAteles paniscus paniscus andAteles paniscus chamek

We studied 20 electrophoretic loci in two populations ofAteles (Ateles paniscus paniscus andAteles paniscus chamek). We observed intrapopulational variation at the following loci: esterase D, glyoxalase 1, adenosine deaminase (A. p. chamek) and carbonic anhydrase 2 (A. p. paniscus). The two populations share the most frequent alleles at 17 loci, but we noted great differences in glyoxalase 1, adenosine deaminase and phosphoglucomutase 1.A. p. paniscus is monomorphic for theGLO1 ^*1 allele, which has a frequency of 6% inA. p.chamek. They did not share alleles in relation to the ADA and PGM1 loci. We found a CA2 allele, named hereCA2 ^*1, which has not been described previously in other neotropical primates (Sampaio et al., 1991a), inA. p. paniscus. The present results suggest that the geographical isolation represented by the Rio Amazonas has lasted long enough to support this level of divergence. These observations taken together with chromosomal findings, led us to endorse the proposal of two distinct species:Ateles paniscus andAteles chamek.     

Genetic mapping and protein product diversity of the self-incompatibility locus in wild tomato (Lycopersicon peruvianum)

Phenotypic diversity of self-incompatibility (S) alleles within nine natural populations ofLycopersicon peruvianum was investigated. Only 7 incompatible responses were observed of a total of 276 unique combinations tested, on the basis of controlled pollinations, indicating the large number of alleles that exist within these populations. Molecular weight polymorphism for specific major stylar proteins observed on SDS-PAGE was also evident in two of the populations examined. Five proteins were shown to map to theS locus and to be associated with differentS alleles through controlled pollinations and segregation of the proteins. Two of theseS related proteins had been described previously in terms of spatial and temporal expression consistent with their involvement in self-incompatibility (Mauet al., Planta 169, 184–191, 1986). A mapping population derived from a fully compatible cross was used to establish linkage of theS locus to two DNA markers,CD15 andTG184, that lie on chromosome 1. The order of the markers and estimates of map distances are given.     

Expressions of GRP78 and Bax associate with differentiation,metastasis, and apoptosis in non-small cell lung cancer

The purpose of this study was to detect the expressions of GRP78 and Bax in human non-small cell lung cancer (NSCLC) tissues, to analyze their correlations with carcinogenesis and the development of NSCLC, and to investigate the relationship of GRP78 expression to metastasis and apoptosis in the NSCLC cell line HCC827. The positive expression rates of GRP78 and Bax in NSCLC lung tissues were 59.7% and 34.7% by RT-PCR, respectively. The mRNA and protein expression levels of GRP78 in NSCLC tissues were significantly higher than that in the relatively normal surrounding lung tissues (p < 0.05); the lesser the degree of tumor differentiation was, the higher the mRNA and protein expression levels of GRP78 were (p < 0.05). The mRNA and protein expression levels of GRP78 from patients in advanced pathological stages (III–IV) were significantly higher than the corresponding levels in patients in early pathological stages (I–II) (p < 0.05); the mRNA and protein expression levels of GRP78 in patients with positive lymph node metastasis were significantly higher than those in patients with negative lymph node metastasis (p < 0.05). The mRNA and protein expression levels of Bax in the above cases showed the opposite trend of the mRNA and protein expression levels of GRP78. However, the mRNA and protein expression levels of both GRP78 and Bax were independent of the patient’s sex, the patient’s age, the tumor size and the histological type (adenocarcinoma or squamous cell carcinoma) of NSCLC (p > 0.05). The mRNA expression level of GRP78 and the mRNA expression level of Bax in human NSCLC tissues were negatively correlated (r = −0.353, p = 0.002). After transfection of GRP78 siRNA in HCC827 cells, the GRP78 protein expression level was significantly decreased (p < 0.01), while the Bax protein expression level was significantly increased (p < 0.01); the number of cells that passed through the Transwell chamber was significantly less in the non-transfected control group compared to the transfected control group (p < 0.01). The number of apoptotic cells was significantly greater in the non-transfected control group compared to the transfected control group (p < 0.01). The expression levels of GRP78 and Bax were related to the carcinogenesis, development and metastasis of NSCLC. GRP78 expression with siRNA interference in the human NSCLC cell line HCC827 can reduce metastasis and promote apoptosis in HCC827 cells.     

Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and of a homologous moiety essential for function

Predicted protein sequences of fungal chitin synthases can be divided into a non-homologous N-terminal region and a C-terminal region that shows significant homology among the various synthases. We have explored the function of these domains by constructing a series of nested deletions, extending from either end, in theCHS1 andCHS2 genes ofSaccharomyces cerevisiae. In both cases, most or all of the sequences encoding the non-homologous N-terminal region (one-third of the protein for Chs1p and about one-fourth for Chs2p) could be excised, with little effect on the enzymatic activity in vitro of the corresponding synthase or on its function in vivo. However, further small deletions (20–25 amino acids) into the homologous region were deleterious to enzymatic activity and function, and often led to changes in the zymogenic character of the enzymes. Similarly, relatively small (about 75 amino acids) deletions from the C-terminus resulted in loss of enzymatic activity and function of both synthases. Thus, it appears that all the information necessary for membrane localization, enzymatic activity and function resides in the homologous regions of Chs1p and Chs2p, a situation that may also apply to other chitin synthases.These authors contributed equally to this paper