Lack of a BglII site at the 5′ region of the PGK 1 locus: a new variant discovered in two Chibchan Amerindian groups from Costa Rica

A new 3.8-kb allele at the 5 region of the PGK 1 locus detected by the probe pSPT/PGK is reported. This variant was discovered in the Cabecar and Guaymi, two Chibchan Amerindian groups of Costa Rica. So far, a polymorphism that consists of an EcoRI/BglI (1.3-kb) variable site within an EcoRI/BglII (1.7-kb) fragment when DNA is simultaneously digested with EcoRI, BglI and BglII is known to occur in black and Caucasian populations. These two alleles were also found in the Amerindians tested. The newly described band is due to the lack of the BglII site situated 1.7 kb downstream from the EcoRI site and to the cleavage of another BglII site 2.1 kb downstream from the lacking one. This variant might be restricted to some Amerindian groups and perhaps also to Asiatic populations. Thus, it could be a useful marker in evolutive studies and for forensic applications. Moreover, the presence of a third allele in populations with Amerindian ancestry can increase the heterozygosity of the region disclosed by the pSPT/PGK probe, thus improving its application in issues dealing with X-chromosome activation ratios in females.     

Characterization of a genomic clone of Erwinia carotovora subsp. carotovora TG3 encoding a pectolytic enzyme of apparent molecular weight 78 kDa

Summary A clone containing the gene encoding a pectolytic enzyme of Erwinia carotovora subsp. carotovora was selected as the one that showed maceration on a solid medium containing sodium polypectate. The gene was located on a 3.2-kb DNA fragment flanked by a BglII site and a Hin-dIII site. Via mini-Mudlac mutagenesis, a possible promoter site was located within the gene between the BglII site and the EcoRI site. The mRNA transcribed from the promoter was directed from the BglII site toward the EcoRI site, determined from the orientation of the inserted mini-Mudlac. The probable gene product was identified as a 78 kDa protein. The enzyme activity of the Escherichia coli clone was detected mainly in the periplasmic space. Potato tuber slices were not macerated by the E. coli clone and synthesis of the enzyme in E. coli was not regulated by the enzyme substrate, sodium polypectate.     

A variation in the structure of the protein-coding region of the human p53 gene

An extensive analysis of genomic DNA preparations from a number of normal and malignant tissues revealed BglII site polymorphism of the human p53 gene. Approximately 10% of p53 gene alleles were found to contain an additional BglII site localized in a region of intron I. This allelic form of p53 gene was also responsible for p53 protein having altered electrophoretic mobility. Molecular cloning and sequencing of both the alleles of p53 gene revealed a base-pair change in codon 72 causing arginine → proline substitution in the allele with the additional BglII site. Both variants of the p53 gene may occur in homozygous state and are therefore functional.     

Isolation of Hem3 mutants from Candida albicans by sequential gene disruption

Summary Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a 2.0 kb fragment by subcloning and a BglII site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the BglII site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura⁺, Leu⁺ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.     

A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance.

Summary This paper reports a cleavage site map of Tn5 for restriction enzymes BamHI, BglI, BglII, HindII, HindIII, HpaI, SalI, AvaI, SmaI, XhoI, PstI, PvuII, HaeII and HaeIII that was determined by the analysis of restriction enzyme cleavage patterns of ColEl, two independent ColE1::Tn5 plasmids, and a ColE1::Tn5 deletion derivative. BalI, EcoRI, KpnI, and PvuI do not cleave Tn5. Construction and analysis of in vitro-generated deletions of a ColE1::Tn5 plasmid limit the sequences encoding neomycin resistance to a 1500-base-pair-long segment of Tn5. Insertion of DNA at a BglII site within this segment results in loss of the neomycin resistance phenotype. Since this BglII site lies in an inverted repeat region, sequences within this repeat seem to be involved in the expression of neomycin resistance.     

DNA polymorphisms in the ε- and γ-globin gene regions in asian macaques

DNA polymorphisms in the ε- and γ-globin gene regions in nine Asian macaques(Macaca fuscata, M. mulatta, M. nemestrina, M. cyclopis, M. fascicularis, M. arctoides, M. radiata, M. maura, andM. assamensis) were examined using several different restriction endonucleases and the human ε and γIVS2 probes.M. fuscata andM. mulatta had highly polymorphic sites(BglII andHincII, respectively) in the ε-globin gene region. TheBamHI site in the γ-globin gene region was highly polymorphic inM. mulatta, while theBglII andHindIII sites in the γ-globin gene region were highly polymorphic inM. fuscata andM. mulatta. The presence of the γ-globin gene triplication was frequently observed inM. fuscata. The gain or loss of an additionalBglII site between two γ-globin genes in Asian macaques seems to be very meaningful for discussing genetic relationships among them. This study was supported in part by the Cooperation Research Program of the Primate Research Institute, Kyoto University.     

Expression of mouse α-amylase gene in methylotrophic yeastPichia pastoris

The expression of the mouse α-amylase gene in the methylotrophic yeast,P. pastoris was investigated. The mouse α-amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested withSalI orBglII, and was introduced intoP. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested withSalI orBglII into theHIS ₄ locus (38 of Mut⁺ clone) or into theAOX1 locus (45 of Mut^s clone). Southern blot was carried out in 11 transformants, which showed that the mouse α-amylase gene was integrated into thePichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α-amylase activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α-amylase gene is compared with that in recombinantSaccharomyces cerevisiae harboring a plasmid encoding the same mouse α-amylase gene, the specific enzyme activity is eight fold higher than that of the recombinantS. cerevisiae.     

Study of restriction fragment length polymorphisms at the human phenylalamine hydroxylase locus and evaluation of its potential application in prenatal diagnosis of phenylketonuria in Chinese

Summary Using a human phenylalanine hydroxylase cDNA probe, the restriction fragment length polymorphisms at the human phenylalanine hydroxylase locus have been determined with the restriction enzymes BglII, PvuII, EcoRI+BamHI, MspI, XmnI, HindIII and EcoRV. The frequency of the observed heterozygosity of the restriction site polymorphisms at this locus in a Chinese population is approximately 54%, which is significantly lower than that in Caucasians. No DNA rearrangement or deletion of the phenylalanine hydroxylase locus was detected among mutant phenylalanine hydroxylase genes in seven Chinese classical phenylketonuria (PKU) families. Haplotype analysis of these seven families revealed that the mutant alleles belonged to five different haplotypes, i.e. haplotype 4, 11 and three unreported haplotypes. The majority of normal and mutant phenylalanine hydroxylase genes are confined to hyplotype 4. These results indicate that approximately 42% of Chinese PKU families are informative for prenatal diagnosis of PKU when eight restriction sites linked to the phenylalanine hydroxylase locus are examined.     

Detection of frequent BglII polymorphism by polymerase chain reaction and TaqI restriction fragment length polymorphism for 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase at the human HSDβ3 locus (1p11–p13)

Summary Analysis of amplified polymerase chain reaction products of 575 bp from the fourth exon of the human type I 3-hydroxysteroid dehydrogenase/⁵-⁴ isomerase gene at locus HSD3 1p11–p13, reveals a frequent two-allele polymorphism at codon Leu³³⁸ due to a silent substitution of T by C, thus creating a BglII site leading to 371- and 204-bp fragments. Southern blot analysis of BglII-digested DNA from 57 individuals using a genomic probe detects two allelic fragments of 5.3kb and 0.77 kb, respectively, while two allelic fragments of 3.7 kb and 3.4 kb are obtained in TaqI digests with multiple constant bands, as also observed with BglII digests.     

A physical map of the permuted genome of bacteriophage T1

Summary A restriction map has been constructed for the DNA of coliphage T1 which locates the cleavage sites of the restriction endonucleases, BglI (6 cuts), BglII (16 cuts), EcoRI (2 cuts), HindIII (2 cuts) and PstI (2 cuts). Digestions with BglI and BglII reveal fragments which are present in sub-molar quantities. Two methods, one using the selective removal of molecular ends with exonuclease III and the other involving the comparison of digestion patterns of concatemeric and virion DNA, have shown that the submolar fragments are at or close to the ends of the molecules. Digestions with BglI show that one terminal fragment has a very precise molecular weight whereas all the others are of heterogenous molecular weight. These results are consistent with the model for DNA packaging in which maturation is initiated at a precise site on a concatemeric precursor and proceeds by the encapsidation of up to four successive headfuls of 1.065 genome equivalents (MacHattie and Gill 1977).     

Cloning and analysis of pif, replication and leading regions of the F plasmid

Summary We describe the molecular cloning of BglII fragments of the hybrid plasmid pRS5 (pSC101 and EcoRI fragments of F; f7, f5, f3 and f6). The clones isolated were examined for the expression of F-specified replication, incompatibility, mobilization and inhibition of T7 bacteriophage multiplication. Proteins directed by the BglII clones were labelled in Escherichia coli K12 maxicells and analyzed by SDS-polyacrylamide gel electrophoresis. The sizes of previously reported proteins, encoded by the replication, incompatibility and leading regions encompassed by these plasmids have been confirmed in this study. In addition, the results demonstrate that a pif gene, which encodes an 80,000 dalton polypeptide essential for the inhibition T7 phage multiplication, is located on the BglII fragment that spans the junction of EcoRI fragments f7 and f5.     

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3 end of the element on germinal excision frequencies. Alteration of three bases to create a BglII site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.     

Genep73: Deletions and expression in non-small-cell lung carcinoma cells

We studied the relation between genetic anomalies in thep73 gene encoding a product structurally and functionally similar to the protein p53 and the pathogenesis of non-small-cell lung carcinoma (NSCLC; 83 patients). Loss of one of thep73 alleles was revealed in 44% (15/34) informative cases. Presence of deletions correlated with the features common for tumor development: metastatic affection of regional lymph nodes (p=0.045), large tumor size (p=0.037), advanced stage of the disease (p=0.017). Allele expression of the genep73 was studied basing on analysis of the polymorphic C/T site in the second exon. All ten studied samples of normal bronchogenic epithelium showed monoallelic expression ofp73, contrary to the six NSCLC samples which preserved both of thep73 alleles and showed biallelic expression. Enhanced expression of p73 mRNA in tumor tissue compared with normal bronchogenic epithelium was found in 28 of 34 (82.4%) NSCLC patients. Expression of p73 in NSCLC cells showed correlation neither with deletions of one of the alleles, nor with any parameter reflecting clinical pathology. The results suggest thatp73 is not a classical tumor suppressor gene in NSCLC. However, alterations ofp73 expression are common for NSCLC. This may be important for our understanding of the NSCLC origin and development.     

The Presence/Absence Polymorphism and Evolution of the p53 Pseudogene in the GenusMus

Distribution of the p53 pseudogene within the house mouse species (genusMus) was studied with polymerase chain reaction for 37 individuals that were caught at different localities. Pseudogene-specific fragments were detected in some, but not all, individuals ofMus musculussubspecies regardless of locality and type of subspecies. In addition, 3 of 7 individuals belonging to differentMusspecies carried the pseudogene in their genomes. These results show the existence of an interspecific presence/absence polymorphism of the p53 pseudogene in mice. Sequence analysis of 11 amplified 0.3-kb fragments suggested that the pseudogene originated in an ancestral mouse about 7 million years ago. Thus alleles with and without the p53 pseudogene have persisted through the mice speciation. The evolutionary rate for the p53 functional gene was also estimated to be about 3.3 × 10⁻⁹per nucleotide site per year.     

Size and organization of a multigene family encoding phaseolin, the major seed storage protein of Phaseolus vulgaris L.

Summary Solution hybridization kinetics and genomic nitrocellulose blot hybridization analyses show that the Phaseolus vulgaris L. (French bean) storage proteins (phaseolins) are encoded as a small, homologous, multigene family consisting of approximately seven members. Restriction endonuclease site mapping (EcoRI, BamHI, and BglII) of DNA regions flanking the phaseolin genes has shown that the gene family can be divided into at least three characteristic fragment size classes. Clones representative of two of these phaseolin gene classes have been isolated from a 1059 phage library.     

Characterization of plQ5 plasmid originating fromKlebsiella pneumoniae

plQ5 plasmid consists of a group of genes specifying resistance to ampicillin, chloramphenicol, carbencillin, kanamycin and trimethoprim-sulphamethoxazole. It is isolated inKlebslella pneumoniae ZD532, is about 26.8 Kb and is freely transmissible to various bacterial species of Gram-negative bacteria. Physical characterization revealed that plQ5 plasmid has a single site forHindill,BamHI,EcoRI and two sites forBglII restriction enzyme.

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Résumé Le blasmide plQ5 consiste en un groupe de genes condant pour la résistance à l'ampicilline, au chloramphénicol, à la carbencilline, à la kanamycine et au triméthoprimsulphamethoxazole. Il est isolé dansKlebsiella pneumoniae ZD532; sa longueur est d'environ 26.8 kb et il est librement transmissible à diverses espèces bactériennes Gram-négatives. La caractérisation physique révèle que le plasmide plQ5 a un site unique pour les enzymes de restrictionHindill,BamHI,EcoRI et deux sites pourBglII.

     

The ORF encoding a putative ferredoxin-like protein downstream of the vnfH gene in Azotobacter vinelandii is involved in the vanadium-dependent alternative pathway of nitrogen fixation

Summary An open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vinelandii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent alternative) pathway of nitrogen fixation was investigated. Site-directed mutagenesis was used to alter one base in each of the codons specifying amino acids 18 and 19 generating a unique BglII site. A kanamycin resistance cartridge was cloned into the BglII site. This construct was mobilized into A. vinelandii CA12 ( nifHDK) strain by conjugation and the mutation was introduced into the genome by marker exchange. The resulting mutant was unable to fix nitrogen under conditions in which the vnf pathway of nitrogen fixation operates. This suggests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitrogen fixation in A. vinelandii.     

Genetic and molecular analysis of familial isolated growth hormone deficiency

Familial isolated growth hormone deficiency (IGHD) has been associated with complete deletions of the hGH-N gene encoding the pituitary growth hormone (GH) in a large number of cases. However, there is still no alternative empirical explanation for the remaining familial or non-familial IGHD cases. We studied a large kindred including five IGHD-affected first cousins to determine possible IGHD inheritance and whether the hGH-N gene was the cause of IGHD in this pedigree. Sex-linked and autosomal recessive transmission of IGHD in this kindred was rejected. Autosomal dominant inheritance was the most probable explanation according to a model of one locus with two alleles, one being dominant for IGHD, under genetic modifiers or epistasis. Southern blotting analysis (BamHI and HindIII digestions) was used to determine whether the hGH-N gene was present in the patients and their family members. Because we found that the hGH-N gene was present, five restriction fragment length polymorphisms (RFLPs; HincII, MspI-A and B, and BglII-A and B) linked to the hGH-N gene were used to try to identify the possible RFLP haplotypes in the pedigree that could be markers or associated with the abnormal hGH-N alleles responsible for IGHD. From the haplotype analysis, it appeared that other genes not linked to the hGH-N gene cluster were the cause of the IGHD phenotype in this kindred. An alternative conclusion could be that the hGH-N gene was responsible for IGHD in this kindred, if a mutation (gene conversion) at the MspI-B site or a reciprocal recombination event between the HincII and MspI-B sites occurred from generation P to F1 and a similar event took place from generation F1 to F2. The non-significant GH responses of patients to the growth releasing factor test confirmed that the hGH-N gene structural product or some step in its regulation was responsible for causing IGHD in this kindred. We suggest that genetic micromutations in the hGH-N gene are present and are responsible for IGHD. We developed a method using the polymerase chain reaction to amplify a 790-bp fragment of the hGH-N gene. The fragment spanned from the second part of the dyad symmetry region in the non-transcribed 5 end of the hGH-N gene to 9 bp before the alternative splice-acceptor site in exon 3. The expected fragment was verified by its digestion with seven diagnostic resctriction endonucleases (BamHI, FspI, PstI, NdeI, BssHII, BglII and HincII). The results showed no deletions or insertions greater than 35 bp in the hGH-N amplified fragment from the DNAs of the IGHD patients and their family members.Presented, in part, at the VIth International Congress of Auxology, Madrid, Spain, 15–19 September 1991.