Partial Diploid Strains Research Articles

Molecular Characterization of Naturally Occurring RNA1 Recombinants of the Comovirus Bean pod mottle virus

ABSTRACT The Bean pod mottle virus, a member of the genus Comovirus, has a bipartite genome consisting of RNA1 and RNA2. We previously reported the occurrence in nature of two distinct subgroups of BPMV strains (subgroups I and II), as well as reassortants between the two subgroups. Here, we report on the isolation and molecular characterization of RNA1 recombinants from soybean plants infected with the partial diploid reassortant strain IL-Cb1, which induces very severe symptoms on soybean. cDNA cloning and sequencing of RNA1 from strain IL-Cb1 revealed the presence of chimeric and mosaic recombinant RNA1s. The full-length mosaic and chimeric recombinant RNA1s were infectious and induced mild symptoms on soybean. Although the recombinant RNA1 accumulated to high levels in the absence of wild-type RNA1, its accumulation level was low in mixed infections with wild-type RNA1. Recombinant RNA1 molecules with similar structures to the naturally occurring recombinant RNA1s were generated in soybean after four passages following inoculation with RNA1 transcripts derived from cDNAs of two distinct strains. This suggests that recombination events are frequent and that a recombination hot spot exists. Sequence analysis of the recombination region showed that it has AU-rich sequences characteristic of recombination hot spots.

Novel Naturally Occurring Bean pod mottle virus Reassortants with Mixed Heterologous RNA1 Genomes

ABSTRACT The comovirus Bean pod mottle virus (BPMV) is widespread in the soybean-growing regions in the United States. It has a bipartite genome consisting of RNA1 and RNA2, which are encapsidated separately. We previously have reported the occurrence in nature of two distinct subgroups of BPMV strains (subgroups I and II), as well as reassortants between the two subgroups. Here, we report the isolation and molecular characterization of naturally occurring partial diploid reassortant strains, which are diploid for RNA1 and haploid for RNA2. Whereas the RNA1s of the partial diploids are derived from two distinct strain subgroups (I and II), the RNA2 is derived from either subgroup I or II. The partial diploid strains induced strikingly severe symptoms on soybean, which could be explained based on the presence of two distinct RNA1s in the same plant. This conclusion was supported by the finding that pseudo-recombinants constructed with two diverse RNA1s induced very severe symptoms on soybean that mimicked those produced by the naturally occurring partial diploids. No enhancement of symptom severity was observed with pseudorecombinants constructed with closely related RNA1s. Likewise, no enhancement of symptom severity was noted with pseudo-recombinants that are diploid for RNA2 and haploid for RNA1. The potential role of genetic reassortment in the epidemiology and pathogenesis of BPMV is discussed.

Microscopic and Ultrastructural Examination of Vegetative Incompatibility in Partial Diploids Heterozygous athetLoci inNeurospora crassa

Vegetative (or heterokaryon) incompatibility is often characterized by cell death after anastomosis. InNeurospora crassa,partial diploid strains heterozygous for a single heterokaryon incompatibility (het) gene are viable, but grow at a significantly inhibited rate. Strains heterozygous forhet-6orhet-cwere examined microscopically for evidence of cell death; ≈15% of cells randomly distributed within such colonies were dead or dying. Electron microscopy revealed extensive organelle degradation and plasmolysis. Ultimately, the cytoplasm fragmented into small membrane-bound bodies. Hyphal regrowth into dying cells from adjacent healthy cells was common. Ultrastructure and cell size measurements indicated no differences in death processes between incompatibility caused byhet-6andhet-c.Linear growth rate was the only measured parameter which correlated with the observed macroscopic differences in colony morphology betweenhetgenes. The ultrastructural changes in dying cells were consistent with descriptions of apoptosis in plants and animals. However, designating vegetative incompatibility as apoptosis is premature without further study.

6 Chromosome Rearrangements in Neurospora and Other Filamentous Fungi

Knowledge of fungal chromosome rearrangements comes primarily from N. crassa, but important information has also been obtained from A. nidulans and S. macrospora. Rearrangements have been identified in other Sordaria species and in Cochliobolus, Coprinus, Magnaporthe, Podospora, and Ustilago. In Neurospora, heterozygosity for most chromosome rearrangements is signaled by the appearance of unpigmented deficiency ascospores, with frequencies and ascus types that are characteristic of the type of rearrangement. Summary information is provided on each of 355 rearrangements analyzed in N. crassa. These include 262 reciprocal translocations, 31 insertional translocations, 27 quasiterminal translocations, 6 pericentric inversions, 1 intrachromosomal transposition, and numerous complex or cryptic rearrangements. Breakpoints are distributed more or less randomly among the seven chromosomes. Sixty of the rearrangements have readily detected mutant phenotypes, of which half are allelic with known genes. Constitutive mutations at certain positively regulated loci involve rearrangements having one breakpoint in an upstream regulatory region. Of 11 rearrangements that have one breakpoint in or near the NOR, most appear genetically to be terminal but are in fact physically reciprocal. Partial diploid strains can be obtained as recombinant progeny from crosses heterozygous for insertional or quasiterminal rearrangements. Duplications produced in this way precisely define segments that cover more than two thirds of the genome. Duplication-producing rearrangements have many uses, including precise genetic mapping by duplication coverage and alignment of physical and genetic maps. Typically, fertility is greatly reduced in crosses parented by a duplication strain. The finding that genes within the duplicated segment have undergone RIP mutation in some of the surviving progeny suggests that RIP may be responsible for the infertility. Meiotically generated recessive-lethal segmental deficiencies can be rescued in heterokaryons. New rearrangements are found in 10% or more of strains in which transforming DNA has been stably integrated. Electrophoretic separation of rearranged chromosomal DNAs has found useful applications. Synaptic adjustment occurs in inversion heterozygotes, leading progressively to nonhomologous association of synaptonemal complex lateral elements, transforming loop pairing into linear pairing. Transvection has been demonstrated in Neurospora. Beginnings have been made in constructing effective balancers. Experience has increased our understanding of several phenomena that may complicate analysis. With some rearrangements, nondisjunction of centromeres from reciprocal translocation quadrivalents results in 3:1 segregation and produces asci with four deficiency ascospores that occupy diagnostic positions in linear asci. Three-to-one segregation is most frequent when breakpoints are near centromeres. With some rearrangements, inviable deficiency ascospores become pigmented. Diagnosis must then depend on ascospore viability. In crosses between highly inbred strains, analysis may be handicapped by random ascospore abortion. This is minimized by using noninbred strains as testers.

Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli

The genes for ribosomal proteins L4 and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the L4 mutation was found to be cold sensitive for growth at 20 degrees C, and 50S-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.

Analysis of Streptococcus pyogenes promoters by using novel Tn916-based shuttle vectors for the construction of transcriptional fusions to chloramphenicol acetyltransferase

We have developed a series of shuttle vectors based on the conjugative transposon Tn916 that have been designed for the analysis of transcriptional regulation in Streptococcus pyogenes and other gram-positive bacteria. Designated the pVIT vectors (vectors for integration into Tn916), the vectors are small, stable plasmids in Escherichia coli to facilitate the fusion of promoters from cloned S. pyogenes genes to a promoterless gene which encodes chloramphenicol acetyltransferase. The vectors each contain one or more small regions of Tn916 to direct the integration of the transcriptional fusion into the transposon via homologous recombination following transformation of S. pyogenes or other suitable gram-positive hosts. Integration can be monitored by the inactivation or replacement of an antibiotic resistance determinant in modified derivatives of Tn916. Promoter activity can then be quantitated by the determination of chloramphenicol acetyltransferase-specific activity. In addition, since integration is into loci that do not disrupt the conjugative transpositional functions of Tn916, the vectors are useful for analysis of regulation in strains that are difficult or impossible to transform and can be introduced into these strains by conjugation following transformation of an intermediate host. The promoters for the genes which encode both the M protein and protein F of S. pyogenes were active in pVIT vectors, as was the region which controls transcription of mry, a trans-acting positive regulator of M protein expression. However, neither of the two characterized promoters for mry demonstrated activity when independently analyzed in pVIT-generated partial diploid strains, suggesting that regulation of mry is more complex than predicted by current models. The broad host range of Tn916 should make the pVIT vectors useful for analysis of regulation in numerous other bacterial species.

Limited differential mRNA inactivation in the atp (unc) operon of Escherichia coli

Individual subunits of ATP synthase, encoded by the eight genes of the atp operon (atpA through atpH), have been found to be synthesized at a 10-fold range in molar amounts (D.L. Foster and R.H. Fillingame, J. Biol. Chem. 257:2009-2015, 1982; K. von Meyenburg, B.B. Jorgensen, J. Nielsen, F.G. Hansen, and O. Michelsen. Tokai J. Exp. Clin. Med. 7:23-31, 1982). We have determined the functional half-lives at 30 degrees C of mRNAs transcribed from these genes either during constitutive expression in a partial diploid strain or after induced expression from a plasmid. Accurate decay kinetics of the relative mRNA levels were determined by monitoring the rates of synthesis of the individual ATP synthase subunits by radioactive pulse labeling at different times after blocking transcription initiation with rifampin. The mRNA transcribed from the atp operon was found to be inactivated about twice as fast as the bulk mRNA in E. coli. Exceptions are the mRNA from the promoter-proximal atpB gene, which was inactivated about three times as fast as the bulk mRNA, and atpC mRNA, the inactivation rate of which was comparable to that of the bulk mRNA. These moderate differences in the kinetics of functional decay explain only a minor part of the differences in expression levels of the atp genes. We conclude, therefore, that the individual atp mRNAs must be translated with widely different efficiencies. The present analysis further revealed that mRNA degradation is sensitive to heat shock; i.e., after incubation at 39 degrees C for 5 min followed by a shift back to 30 degrees C, the decay rate of the bulk mRNA was decreased by 30%.

Ribosomal DNA is a site of chromosome breakage in aneuploid strains of Neurospora.

In wild-type strains of Neurospora crassa, the rDNA is located at a single site in the genome called the nucleolus organizer region (NOR), which forms a terminal segment on linkage group (LG) V. In the quasiterminal translocation strain T(I;V)AR190, most of the right arm of LG I moved to the distal tip of the NOR, and one or a few rDNA repeat units are moved to the truncated right arm of LG I. I report here that, in partial diploid strains derived from T(I;V)AR190, large terminal deletions result from chromosome breakage in the NOR. In most of these partial diploids, chromosome breakage is apparently frequent and the breakpoints occur in many parts of the NOR. The rDNA ends resulting from chromosome breakage are "healed" by the addition of new telomeres. Significantly, the presence of ectopic rDNA creates a new site of chromosome breakage in the genome of partial diploids. These results raise the possibility that, under certain conditions, rDNA is a region of fragility in eukaryotic chromosomes.

Genetic analysis of mutants affected in the Pst inorganic phosphate transport system.

A number of mutant alleles affecting the Pst phosphate transport system have been divided into three complementation groups on the basis of constitutive alkaline phosphatase activity in appropriate partial diploid strains. The three complementation groups were represented by the alleles pstA2 and phoT32 and the newly described allele pstB401. The two alleles phoS28 and phoS21 appeared to be polar. The phoS28 allele affected both the phoT and pstB genes but not the pstA gene, whereas the phoS21 allele appeared to be a mutation in the pstA gene exerting polar effects on both the pstB and phoT genes. It was concluded that the three genes pstA, pstB, and phoT were part of an operon and that the phosphate-binding protein was not coded for by any of these genes. The phoS gene, defined as the structural gene for the phosphate-binding protein, is also part of the operon, but the phoS28 and phoS21 alleles are not mutations in the phoS gene and were reclassified as pho-28 and pho-21 alleles. The gene order was concluded to be pstA-(pstB-phoT)-phoS, with the pstA gene promotor proximal and the direction of transcription opposite to that of the nearby unc operon.

Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12

Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme.

A Fifth Gene ( uncE ) in the Operon Concerned with Oxidative Phosphorylation in Escherichia coli

Three mutant unc alleles (unc-408, unc-410, and unc-429) affecting the coupling of electron transport to oxidative phosphorylation in Escherichia coli K-12 have been characterized. Genetic complementation analyses using previously defined mutant unc alleles indicated that the new mutant unc alleles affect a previously undescribed gene designated uncE. The phenotype of strains carrying the uncE408 or uncE429 allele is similar in that Mg(2+)-adenosine triphosphatase activity is only found in the cytoplasmic fraction, and membranes do not bind the F(1) portion of adenosine triphosphatase purified from a normal strain. In contrast, adenosine triphosphatase activity is present both in the cytoplasm and on the membranes from a strain carrying the unc-410 allele, and normal F(1) binds to F(1)-depleted membranes from this strain. The adenosine triphosphatase solubilized from membranes of a strain carrying the unc-410 allele reconstituted ATP-dependent membrane energization in F(1)-depleted membranes from a normal strain. Genetic complementation tests using various Mu-induced unc alleles in partial diploid strains show that the uncE gene is in the unc operon and that the order of genes is uncB E A D C. The unc-410 allele differs from the uncE408 and uncE429 alleles in that complementation tests with the Mu-induced unc alleles indicate that more than one gene is affected. It is concluded that this is due to a deletion which includes part of the uncE gene and another gene, or genes, between the uncE and uncA genes.

Characterization of the mutant-unc D-gene product in a strain of Escherichia coli K12. An altered beta-subunit of the magnesium ion-stimulated adenosine triphosphatase.

Membranes from a mutant strain of Escherichia coli K12 carrying the uncD409 allele were washed in low-ionic-strength buffers in the presence or absence of the proteinase inhibitor p-aminobenzamidine. Unlike membranes from a normal strain, those from strain AN463 (uncD409) did not become proton-permeable, as judged by NADH-induced atebrinfluorescence quenching, when the membranes were washed in the absence of p-aminobenzamide. Furthermore, ATP-dependent atebrin-fluorscence quenching in such washed membranes could not be reconstituted by the addition of solubilized Mg2+-stimulated adenosine triphosphatase preparations. The examination by two-dimensional polyacrylamide-gel electrophoresis of the polypeptide composition of the washed membranes from strain AN463 (uncD409) indicated the presence of a polypeptide of similar molecular weight to the normal beta-subunit of the Mg2+-stimulated adenosine triphosphatase, but with an altered isoelectric point. Both the normal and abnormal beta-subunits were identified in membranes prepared from a partial diploid strain carrying both the unc+ and uncD409 alleles. It is concluded that the uncD gene codes for the beta-subunit of the Mg2+-stimulated adenosine triphosphatase.

Mu-induced polarity in the unc operon of Escherichia coli.

Mutant strains of Escherichia coli were isolated in which mutator (Mu) phage was inserted into various unc genes. Partial diploid strains were prepared from each of the Mu-induced unc mutants by using F-plasmids carrying mutations in one of the known unc genes (uncA, uncB, uncC, or uncD). The partial diploid strains and the corresponding segregant strains were examined for their ability to grow on succinate. The aerobic growth yields on limiting concentrations of glucose were also determined. Magnesium-stimulated adenosine triphosphatase activities, ATP-dependent transhydrogenase activities, and Atebrin fluorescence quenching activities were determined by using membrane preparations from each strain. Genetic complementation was assessed from the results obtained, and it was concluded that the four unc genes examined are part of a single transcriptional unit and that they are transcribed in the order uncBADC.

Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the Fl portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.

A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase. A method was developed to incorporate mutant unc alleles into plasmids. Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa. Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements. The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424.

Transcription of ribosomal protein genes carried on F′ plasmids ofEscherichia coli

The transcriptional activity of the DNA sequences coding for certain ribosomal proteins has been measured in Escherichia coli. Two partial diploid strains were isolated from mating of a recA, argD, aroE recipient and an Hfr with an origin at 69.5 min. One contained an F' element carrying the genes from 69.5 to 72.7 min including the rpsL locus (72.4) and the second was diploid for the genes from 69.5 to 71.5 min but did not include any mapped ribosomal protein loci. The molecular weights of the plasmids were estimated to be 140 and 110 x 10(6) daltons, respectively. The extent of in vivo transcription of the chromosomal genes on the plasmid and the ribosomal protein mRNA fraction of the total cellular RNA weer calculated from DNA-RNA liquid hybridization experiments using both DNA and RNA excess procedures. The results indicated a high degree of transcriptional activity concentrated in the ribosomal protein sequences with 83% of the F' chromosomal sequences transcribed into mRNA products representing about 0.12% of the total cellular RNA.

Significance of constitutive integrase synthesis.

One conceivable function for constitutive integrase formation by lambda prophage is to stabilize the inserted state by catalyzing reinsertion of prophages that are accidentally excised. As this hypothesis implies a dynamic equilibrium betwen inserted and noninserted DNA, the existence of such an equilibrium is explored. By examining the frequency with which prophages appear in an initially unoccupied chromosomal site of a lysogenic bacterium in which the prophage attachment site is duplicated, the off-rate is estimated as less than 10(-2) per generation for wild-type lambda, and less than 4 x 10(-4) for N- mutants of lambda. From the rate of integrase-catalyzed haploidization of certain partial diploid strains, the rate of spontaneous integrase activity is estimated as 3 x 10(-3) per generation. From these values I conclude that constitutive integrase will not appreciably stabilize the inserted state by virtue of its known activity.

A new relaxed mutant of Escherichia coli with an altered 50S ribosomal subunit.

A new relaxed mutant called rel C has been isolated from a rel A(+)/rel A(+) partial diploid strain. The rel C mutant is unable to synthesize ppGpp or pppGpp in vivo in response to amino acid starvation or in vitro, but can synthesize these nucleotides in a shift-down. Rel C maps near rif. Studies in vitro demonstrate the lesion to be probably in one of the 50S ribosomal proteins that can be removed by 1.0 M LiCl.

Genetic analysis of bacteriophage Mu-induced flagellar mutants in Escherichia coli.

In previous work, at least 10 discrete cistrons involved in the synthesis of flagella in Escherichia coli were described. Six cistrons were located between his and uvrC on the genetic map. These were referred to as hag, flaA, flaB, flaC, flaD, and flaE. Four cistrons referred to as mot, flaG, flaH, and flaI were located between uvrC and aroD. In order to determine whether these genes are organized into transcriptional units, a series of Mu phage-induced flagellar mutants was studied. The mutant strains behaved as if they were carrying strong polar mutations. Of 228 independent Mu-induced mutants, 114 with mutations in the his-aroD region of the genetic map were tested by preparing partial diploid strains with episomes carrying a variety of previously defined mutations. The pattern of complementation that emerged indicated that cistrons flaB, flaC, and flaE form a transcriptional unit. Cistron flaO, defined in the course of this study, is also a member of this transcriptional unit. The order of transcription is B-C-O-E. flaA was found to be complex, and it included four cistrons, flaA, flaP, flaQ, and flaR, with the transcriptional order A-P-Q-R. Cistrons flaG and flaH are cotranscribed with the transcriptional order G-H. The remaining genes, flaD, flaI, hag, and mot do not belong to multicistronic transcriptional units. Complementation analysis suggested that the cheC locus is the same as cistron flaA.

Properties of a temperature sensitive plasmid in Citrobacter freundii.

The properties of a strain of Citrobacter freundii with a deletion of the gal, chl D, bio, uvr B, chl A region of the chromosome, harbouring a temperature sensitive plasmid F ts 114 1 aro G+ gal + chl D+ bio + uvr B+, originating from Escherichia coli, are described. The isolation of strains with an integrated plasmid was tried by incubation of the partial diploid strain at the restrictive temperature and selection for retained plasmid properties. However C. freundii Hfr strains were not obtained, since only fragments of the plasmid were integrated. Integration occurred at seven different sites of the chromosome and resulted in an inactivation of the gene in which the fragment was integrated. Mutants with deficiencies for arginine, isoleucine and valine, tryptophan, guanine and either tryptophan or tyrosine were obtained. In another type the deficiency, resulting from integration, could not be identified, whereas in the seventh type integration had occurred in one or more non-essential genes, because no deficiency was present. Release of the integrated fragment occurred in such a way that gene activity was restored. The released fragment was lost or was integrated again at one of the other six integration sites, resulting in another mutant type.