A homologous and self-replicating system for efficient transformation ofFusarium oxysporum

A highly efficient transformation system has been developed forFusarium oxysporum f. sp.lycopersici based on the complementation of a nitrate-reductase mutant with the homologousnitI gene and on the presence ofARS and telomeric sequences in the vector. Preliminary transformation experiments with theniaD gene fromAspergillus niger generated self-replicating plasmids within the transformed entity that contained extra-fungal DNA. A fragment of the extra DNA was inserted into pUC19 together with theF. oxysporum nitl gene, resulting in plasmid pFNit-Lam. This allowed the isolation of a new linear plasmid within self-replicativeF. oxysporum transformants (pFNit-Lam-TLam, linear). The circular form of this vector yielded 5600 fungal transformants per g of DNA. All of the transformants contained autonomous linear plasmids harboring direct repeats of fungal DNA at both ends. The sequence of the 1.2-kb fragment fromF. oxysporum responsible for autonomous replication, and maintenance as linear plasmid molecules, has been determined. Comparison analysis with theARS from different organisms has shown that this fragment contained the commonly identifiedARS consensus sequence, 5A/TTTTATA/GTTTA/T3 and, in addition to this core, ten copies of theARS-box, TNTA/GAA3. Adjacent to this presumedARS, the telomeric hexanucleotide sequence (TTAGGG)_n was present in six tandem copies followed by 18 copies of its complementary sequence.     

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

Summary A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5-phosphate-decarboxylase gene. A. niger Pyr^– mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/g DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA⁺ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.     

Expression of an Erwinia pectate lyase in three species of Aspergillus

Transgenic filamentous fungi of the speciesAspergillus niger, A. nidulans andA. awamori expressing and secretingErwinia carotovora subsp.atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using theA. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide ofA. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro-sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0 mg/l for expression inA. niger, A. awamori andA. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes.     

Differences in the regulation of aldehyde dehydrogenase genes inAspergillus niger andAspergillus nidulans

Summary In order to study mechanisms of gene regulation inA. niger, and to compare these to similar systems inA. nidulans, a gene encoding an aldehyde dehydrogenase enzyme has been cloned. In wild-type strains ofA. niger the gene shows expression which is regulated by induction and repression. Levels of induction by various compounds and the extent of repression under various growth conditions differs from that seen for theA. nidulans aldA gene. Unlike the A.nidulans aldA gene, theA. niger gene has both carbon catabolite repressible and nonrepressible induction mechanisms. Studies of heterologous expression of theA. niger gene inA. nidulans have shown that its expression is regulated by thealcR gene product ofA. nidulans.     

Ca2+ transport of the skeletal muscle sarcoplasmic reticulum during readaptation of rats after a 40-day load relief on the hind paws

The rate of Ca²⁺ accumulation in the sarcoplasmic reticulum of the skeletal muscle (m. gastrocnemius lateralis, m. vastus medialis, andm. soleus) is studied in rats under conditions of functional off-loading of the hind paws (suspending animals by the tail). The rate of Ca²⁺ transport in the sarcoplasmic reticulum is shown to be stepped up in all these muscles. In the sarcoplasmic reticulum ofm. gastrocnemius lateralis andm. vastus medialis the Ca²⁺ transport rate reliably drops, which does not occur inm. soleus. During a 2-week period of readaptation of animals suspended for 40 days, the Ca²⁺-transporting function of them. soleus sarcoplasmic reticulum gradually recovers to reach the control values, whereas the time course of recovery of Ca²⁺-pump activity in the sarcoplasmic reticulum ofm. gastrocnemius lateralis andm. vastus medialis has a phasic pattern. Translated fromByulleten' Eksperimental'noi Biologii i Meditsity, Vol. 118, N ^o 12, pp. 591–595, December, 1994 Presented by A. I. Grigor'ev, Member of the Russian Academy of Medical Sciences     

Female sexual receptivity is defective in juvenile hormone-deficient mutants of theapterous gene ofDrosophila melanogaster

During reproductive maturation of female insects, the acquisition of sexual receptivity is coordinated with ovarian development. Juvenile homone regulates vitellogenesis in the ovaries, but the action of this hormone in the development of sexual behavior is less well-understood. A strain ofDrosophila melanogaster carrying a mutation in theapterous gene(ap ⁴) was known to exhibit arrested vitellogenesis (rescuable by applying exogenous juvenile hormone), sterility of both sexes, and a deficiency of juvenile hormone. In this study, we examined the effects of mutations ofap on female receptivity and its relationship to juvenile hormone. We observed abnormally low female receptivity in homozygousap strains, and heteroallelic combinations ofap mutations exhibited low receptivity. For female receptivity,ap showed no dominance (i.e.,ap/ap ⁺ was intermediate betweenap/ap andap ⁺/ap ⁺). Low receptivity mapped genetically to theap locus. The reduction in female receptivity in these mutants is positively correlated with levels of juvenile hormone synthesized by their corpora allata.This work was supported in part by The Scheinfeld Center for Humans Genetics in the Social Sciences (J.R.), The National Science Foundation (BNS-882 1339 to J.R.), BARD (No. IS-1664-89R to D.S.), The Israel Cancer Research Fund (grant to D.S.), The Rekanati Foundation of Tel Aviv University (grant to D.S.), and The Israeli Fruit Council (award to M.A.)     

Isolation and characterization of a 1,4-β-endoxylanase gene of A. awamori

An enzyme with a particular 1,4--xylanase activity was identified and purified from wheat-bran culture medium of an Aspergillus awamori strain. With oligonucleotides based on the N-terminal amino-acid sequence of the enzyme, the exlA gene of A. awamori, encoding 1,4--xylanase A, has been cloned. Based on the deduced amino-acid sequence, 1,4--xylanase A is produced as a 211 amino-acid-residue-long precursor, which is converted post-translationally into a 184-aa-residue-long mature protein. Transformation of the original A. awamori strain with multiple copies of the exlA gene resulted in a 40-fold overproduction of 1,4--xylanase A. The overproduced enzyme has the same biochemical and enzymological properties as the wild-type enzyme.     

Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene

A wild isolate of Penicillium canescens was subjected to mutagenesis, and 150 chlorate-resistant mutants were isolated and classified in respect of their ability to utilize various nitrogen sources. Strains supposedly deficient in nitrate reductase have been transformed with the nitrate-reductase gene from Aspergillus niger. Transformation probably occurred by non-homologous integration of the transforming vector into the chromosome. Co-transformation with the AMA1 replicating element from A. nidulans enhanced transformation frequency up to 2000-fold, and was shown to result in autonomous maintenance of replicating concatenates, one of which was re-isolated by transformation of E. coli.     

Functional nuclear suppressor of mitochondrialoxi2 mutations in yeast

Summary A semidominant nuclear suppressor, callednam6, ofoxi2-V276 mitochondrial mutation has been isolated and characterized. The nuclear character ofnam6 was proved by its retention inrho° strains, lack of mitotic segregation in diploids and meiotic 2:2 segregation in tetrads. The specificity ofnam6 was tested on 315mit ^– mutations of four mitochondrial genes (oxi1, oxi2, oxi3, andcob-box). It suppresses clearly only three mutations in theoxi2 gene, restoring partially or completely cytochrome aa₃ formation. The results suggest a functional character of the suppression.     

Analysis of the first intron ofTNFB gene byNcoI RFLP in koreans

Summary The tumor necrosis factor B (TNFB) gene is closely liked with tumor necrosis factor A (TNFA) gene between theHLA-B andC2 genes on chromosome 6p21.3. Several genetic variabilities at the humanTNFB loci have been identified, which are theNcoI restriction fragment length polymorphism (RFLP) in the first intron, amino acid substitution at codon 26 of exon 3 andEcoRI RFLP in untranslated exon 4. TheNcoI RFLP ofTNFB gene gives two allelic fragments of 238/259 bp and 497 bp, corresponding toTNFB*1 andTNFB*2 alleles, respectively. To investigate the frequency ofNcoI RFLP in the first intron ofTNFB in Koreans and to compare to that of other ethnic population, genomic DNAs were extracted from leukocytes of 305 unrelated healthy Koreans and amplified the first intron ofTNFB gene by PCR. The phenotype frequencies ofNcoI RFLP such asTNFB*1/TNFB*1, TNFB*1/TNFB*2 andTNFB*2/TNFB*2 were 8.6% (n=26), 45.2% (n=138) and 46.2% (n=141), respectively. The estimated allele frequencies forTNFB*1 andTNFB*2 were 0.3115 and 0.6885, respectively. The observed and expected frequencies were in good agreement with the Hardy-Weinberg’s equilibrium. The heterozygosity revealed 45.2% and the allele frequencies ofNcoI RFLP ofTNFB in Koreans were observed comparatively similar to those of other ethnic groups.     

Cloning of a gene associated with aflatoxin B1 biosynthesis in Aspergillus parasiticus

Summary A cosmid library was constructed by inserting genomic DNA isolated from a wild-type aflatoxin-producing strain of Aspergillus parasiticus (SU-1) into a cosmid vector containing an homologous nitrate reductase (niaD) gene as a selectable marker. One cosmid was isolated which complemented an aflatoxin-deficient, nitrate-nonutilizing mutant strain, A. parasiticus B62 (nor-1, niaD), to aflatoxin production. Deletion and complementation analyses showed that, a 1.7 kb BglII-SphI DNA fragment isolated form this cosmid was responsible for renewed aflatoxin production. Northern hybridization analyses revealed that the major RNA transcribed from this DNA fragment, was 1.4 kilonucleotides in size. Genetic complementation, proved to be a useful strategy for cloning a gene associated with aflatoxin biosynthesis in A. parasiticus.     

Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number,growth rate and enzyme activity

Summary nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutaminerich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.     

NcoI restriction fragment length polymorphism at — 308 of the tumor necrosis factor alpha (TNFA) promoter region in korean

Summary Tumor necrosis factor alpha (TNFA) is a cytokine, which is secreted from activated macrophage, with a broad range of biological activities. The gene encodingTNFA is located in tandem with theTNFB gene within the HLA complex on chromosome 6p21.3. We detected a single base polymorphism in the humanTNFA gene promoter region in 300 unrelated Korean individuals. TheTNFA promoter region which showed a G to A transition at position of −308 was investigated byNcoI restriction fragment length polymorphism analysis. A biallelic polymorphism ofTNFA gene showed fragments of 87/20 bp and 107 bp acting asTNFA*1 allele andTNFA*2 allele, respectively. The allele frequencies ofTNFA*1 andTNFA*2 were 0.8783 and 0.1217, respectively. The 21.7% of heterozygosity was observed. No association between promoter region phenotypes ofTNFA and the first intron phenotypes ofTNFB was observed in Korean. Allele frequencies of Koreans were compared with that of Europeans.     

Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): Indications of a pectin lyase gene family in A. niger

Summary Using the previously cloned Aspergillus niger N756 pectin lyase D gene as a probe, the corresponding pelD gene has been isolated from a genomic library of the loboratory strain A. niger N400. This gene encodes PLD, previously described as PLI, which is one of the two major pectin lyases isolated from the commeriial pectinase preparation Ultrazym^®. Heterologous hybridization of the A. niger N400 genomic library with the pelD gene led to the isolation of another five genes: pelA, B, C, E, and F. These genes differ in their hybridization patterns with probes containing either the entire pelD gene, or 5 or 3 parts thereof. By partial sequencing, and expression in an A. niger transformant containing multiple copies of the pelA gene, we show that this gene, which hybridizes strongest with the pelD gene, encodes the other major pectin lyase from Ultrazym^®, PLII.     

Male sexual signaling is defective in mutants of theapterous gene ofDrosophila melanogaster

Theapterous (ap) gene ofDrosophila melanogaster exhibits extreme pleiotrophy: its functioning is essential for life, normal wing structure, juvenile hormone production, female fertility, and normal development of female sexual receptivity. Four mutantap alleles (ap ⁴,ap ^56f,ap ^c, andap ^blt) were characterized for three additional phenotypes: male mating success, courtship behavior, and immature male sex appeal (the ability of males to stimulate homosexual cortship). Mating success with mature wild-type virgin females is reduced in males mutant for theap gene, the extreme case beingap ⁴/ap ⁴ males, which are behaviorally sterile. Inap mutants, nonwing courtship elements are qualitatively like those ofap ⁺/ap ⁺ males. However, the mean rate of nonwing courtship directed toward virgin wild-type females (i.e., the mean temporal frequency of these displays) is reduced in males homozygous forap ⁴,ap ^56f, orap ^c alleles. In contrast, theap ^blt allele makes for wild-type rates of nonwing courtship. Immature male sex appeal persists for at least 3 days in males homozygous forap ^c and, to a lesser extent, inap ^56f orap ⁴ homozygotes;ap ^blt/ap ^blt and wild-type males lose immature male sex appeal after 1 day. All three male phenotypes map to theap locus, which is therefore essential for the development of normal levels of male courtship and male mating success and for the timely loss of immature male sex appeal. For each phenotype,ap ⁺ is dominant toap alleles making for behavioral abnormalities, with a single exception (for rate of nonwing courtship,ap ⁺/ap ^c was low). For mating success and frequency of nonwing courtship, each allele pair exhibits at least partial complementation, except forap ⁴ andap ^56f, which fail to complement. For immature male sex appeal,ap ^c,ap ⁴, andap ^56f fall into the same complementation group. Juvenile hormone production is not correlated with effects on male reproductive behavior.This work was supported in part by The Scheinfeld Center for Human Genetics in the Social Sciences (J.R.), The National Science Foundation (BNS-882 1339, to J.R.), BARD (IS-1664-89R, to D.S.), and The Israel Cancer Research Fund (grant to D.S.).     

Role ofBcl-2, Bax, andBak in spontaneous apoptosis and proliferation in neuroendocrine lung tumors: Immunohistochemical study

Fifty-six primary neuroendocrine lung tumors were examined morphologically and histologically and their apoptosis level was determined. Malignant carcinomas were characterized by increased apoptotic index and enhanced expression ofBcl-2, Bak, p53, andKi-67 compared to typical carcinoid. However, apoptosis in these tumors was not completed. Proteins of theBcl family play an important role in the regulation of spontaneous apoptosis in neuroendocrine lung tumors.Bcl-2 accumulating in the nucleus is a morphological analogue of phosphorylated inactive form of this protein, which does not inhibit apoptosis. Expression ofBcl-2 andBax decreases in small-cell lung carcinoma (SCLC) with metastases indicating attenuation of apoptosis and development of metastatic clones resistant to apoptosis induces. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 7, pp. 98–101, July, 2000     

Specificities of the mechanism of bacterial development during the lag phase

The mechanisms ofE. coli development during the lag phase are described on the basis of biophysical characteristics of the bacterial cell. A bioelectromagnetic model closely approximates the experimental parameters of the time course ofE. coli andE. aerogenes development in the mono- and mixed culture. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o , 3, pp. 331–336, March, 1994 Presented by A. D. Ado, Member of the Russian Academy of Medical Sciences     

Chromosome mapping ofSoa,a gene influencing gustatory sensitivity to sucrose octaacetate in mice

Strain distribution patterns among recombinant inbred strains suggested that a locus influencing taste sensitivity to sucrose octaacetate was on chromosome 6. A location forSoa was established by linkage analysis of behavioral and electrophoretic data from outbred and congenic strains and from test-cross progeny. Haplotyping of 41 outbred CFW-Cr animals with a cDNA probe showed perfect cosegregation ofSoa andPrp, a gene for salivary proline-rich proteins. Five of twelve B6. SW-Soa ^a strains were found to retainLdr-1, lactate dehydrogenase regulator-1, on chromosome 6 as an allelic passenger from the SWR/J donor strain (source of theSoa ^a Taster allele). Centimorgan distance was estimated using the ABP/Le linkage-testing strain (non-Taster,Soa ^b) and the SWR/J strain (Taster,Soa ^a) in a testcross breeding system. The data are consistent with a position for theSoa locus on mouse chromosome 6, 62 cM from the centromere.This research was supported in part by Grants DC00150 (G. W.) and DE003658 (E.A.A.). This paper is based on a thesis submitted to the Florida State University by the first author in partial fulfillment of the requirements for the Master of Sciences degree.     

Role of Bcl-2, Bax, and Bak in spontaneous apoptosis and proliferation in neuroendocrine lung tumors: immunohistochemical study

Fifty-six primary neuroendocrine lung tumors were examined morphologically and histologically and their apoptosis level was determined. Malignant carcinomas were characterized by increased apoptotic index and enhanced expression ofBcl-2, Bak, p53, andKi-67 compared to typical carcinoid. However, apoptosis in these tumors was not completed. Proteins of theBcl family play an important role in the regulation of spontaneous apoptosis in neuroendocrine lung tumors.Bcl-2 accumulating in the nucleus is a morphological analogue of phosphorylated inactive form of this protein, which does not inhibit apoptosis. Expression ofBcl-2 andBax decreases in small-cell lung carcinoma (SCLC) with metastases indicating attenuation of apoptosis and development of metastatic clones resistant to apoptosis induces. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 7, pp. 98–101, July, 2000