The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase.

To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified.     

A Recessive Circadian Clock Mutation at the frq Locus of NEUROSPORA CRASSA

A circadian clock mutant of Neurospora crassa, the most distinctive characteristic of which is the complete loss of temperature compensation of its period length, maps to the frq locus where seven other clock mutants have previously been mapped. This mutant, designated frq-9, is recessive to the wild-type allele and to each of the other frq mutants; thus, it differs from the other mutants, which show incomplete dominance to wild type and to each other. Complementation analysis suggests either that the frq locus is a single gene or that frq-9 is a deletion that overlaps adjacent genes. Preliminary efforts at fine structure mapping have indicated that recombination between certain pairs of frq mutations is less than 0.005%, a distance consistent with the locus being a single gene. The recessive nature of frq-9, coupled with complete loss of temperature compensation, suggests that this mutant may represent the null phenotype of the locus and that the frq gene is involved in the temperature compensation mechanism of the clock.--Genetic mapping studies have placed the frq locus on linkage group VIIR, midway between oli (oligomycin resistance) and for (formate auxotrophy), about 2 map units from each, and clearly indicate that frq and oli are separate genes.     

Mild RIP — an alternative method for in vivo mutagenesis of thealbino-3 gene inNeurospora crassa

We have used a biological phenomenon that occurs inNeurospora crassa, termed Repeat-Induced Point mutation (RIP), to create partially functional mutant alleles of thealbino-3 (al-3) gene encoding geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids and diverse prenylated compounds. A total of 70 RIP-inducedal- 3 mutants were identified by their pale albino phenotype, resulting from inactivation of carotenoid biosynthesis. Nucleotide sequence analysis of theal-3 gene in five of the RIP-induced mutants revealed that in each case RIP had introduced no more than six point mutations. The low frequency of RIP mutants (0.42%) and the isolation of only leaky mutants with very few mutations suggest that ascospores containing a heavily mutatedal-3 gene do not survive. These results are evidence that the RIP phenomenon, used to inactivate and silence duplicated genes inN. crassa, may be exploited in its mild version as a method of sequence-specific in vivo mutagenesis to obtain functional mutant alleles ofNeurospora genes. This mild form of mutagenesis may be particularly advantageous in selecting for leaky mutations in essentialNeurospora genes.     

The Cpx proteins of Escherichia coli K-12: evidence that cpxA, ecfB, ssd, and eup mutations all identify the same gene.

An existing cpxA(Ts) mutant was resistant to amikacin at levels that inhibited completely the growth of a cpxA+ and a cpxA deletion strain and failed to grow as efficiently on exogenous proline. These properties are similar to those of mutants altered in a gene mapped to the cpxA locus and variously designated as ecfB, ssd, and eup. The amikacin resistance phenotype of the cpxA mutant was inseparable by recombination from the cpxA mutant phenotype (inability to grow at 41 degrees C without exogenous isoleucine and valine) and was recessive to the cpxA+ allele of a recombinant plasmid. Using methods that ensured independent mutations in the cpxA region of the chromosome, we isolated six new amikacin-resistant mutants following nitrosoguanidine mutagenesis. Three-factor crosses mapped the mutations to the cpxA locus. When transferred by P1 transduction to a cpxB11 Hfr strain, each of the mutations conferred the Tra- and Ilv- phenotypes characteristic of earlier cpxA mutants. Two of the new mutations led to a significantly impaired ability to utilize exogenous proline, and four led to partial resistance to colicin A. Two of the new cpxA alleles were recessive to the cpxA+ allele, and four were dominant, albeit to different degrees. On the basis of these data, we argue that cpxA, ecfB, eup, and ssd are all the same gene. We discuss the cellular function of the cpxA gene product in that light.     

Identification of the gene at the pmg locus, encoding system II, the general amino acid transporter in Neurospora crassa.

Mutations at the pmg locus in Neurospora crassa cause a deficiency in a transport system for a broad range of amino acids. We have isolated a gene that encodes a protein with a high degree of sequence similarity to the GAP1 general amino acid permease in Saccharomyces cerevisiae. Our data indicate that this is the gene at the pmg locus. It encodes a 572-residue protein with a molecular mass of 62,649 Da. The predicted secondary structure has 12 membrane-spanning regions, a feature characteristic of a superfamily of permease proteins. Inactivation of the gene yielded a mutant strain with the same phenotype as the pmg- strain, and a cosmid containing a functional copy of the gene rescued the pmg- strain. Although the pmg- strain has previously been assayed in a genetic background that contains mutations in genes for two other amino acid transport systems, we have found conditions in which the pmg- strain has an identifiable phenotype in an otherwise wild-type genetic background.     

Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene

We find that two Chlamydomonas outer arm dynein assembly loci, oda6 and oda9, are located on the left arm of linkage group XII, in the vicinity of the previously mapped locus for a 70,000 Mr dynein intermediate chain protein. Restriction fragment length polymorphism mapping indicates that this dynein gene is very closely linked to the oda6 locus. A cDNA clone encoding the 70,000 Mr protein was isolated, sequenced, and used to select genomic clones spanning the corresponding locus from both wild-type and oda6 libraries. When wild-type clones were introduced into cells containing an oda6 allele, the mutant phenotype was rescued, while no rescue was observed after transformation with oda6 clones. Genetic analysis further revealed that newly introduced gene copies were responsible for the rescued phenotype and thus confirms that ODA6 encodes the 70,000 Mr dynein intermediate chain protein. The inability of oda6 mutants to assemble any major outer arm dynein subunits shows that this protein is essential for assembly of stable outer dynein arms. This is the first use of transformation with a wild-type gene to identify the product of a Chlamydomonas mutant.     

Methylation of histone H3 lysine 36 is required for normal development in Neurospora crassa

The SET domain is an evolutionarily conserved domain found predominantly in histone methyltransferases (HMTs). The Neurospora crassa genome includes nine SET domain genes (set-1 through set-9) in addition to dim-5, which encodes a histone H3 lysine 9 HMT required for DNA methylation. We demonstrate that Neurospora set-2 encodes a histone H3 lysine 36 (K36) methyltransferase and that it is essential for normal growth and development. We used repeat induced point mutation to make a set-2 mutant (set-2(RIP1)) with multiple nonsense mutations. Western analyses revealed that the mutant lacks SET-2 protein and K36 methylation. An amino-terminal fragment that includes the AWS, SET, and post-SET domains of SET-2 proved sufficient for K36 HMT activity in vitro. Nucleosomes were better substrates than free histones. The set-2(RIP1) mutant grows slowly, conidiates poorly, and is female sterile. Introducing the wild-type gene into the mutant complemented the defects, confirming that they resulted from loss of set-2 function. We replaced the wild-type histone H3 gene (hH3) with an allele producing a Lys to Leu substitution at position 36 and found that this hH3(K36L) mutant phenocopied the set-2(RIP1) mutant, confirming that the observed defects in growth and development result from inability to methylate K36 of H3. Finally, we used chromatin immunoprecipitation to demonstrate that actively transcribed genes in Neurospora crassa are enriched for H3 methylated at lysines 4 and 36. Taken together, our results suggest that methylation of K36 in Neurospora crassa is essential for normal growth and development.     

Heterokaryon incompatibility in fungi—more than just another way to die

In filamentous fungi heterokaryon (vegetative) compatibility is regulated by a number of different loci. Vegetative incompatibility is most often detected as the inability to form a prototrophic heterokaryon under forcing conditions, or as the formation of a barrage when two incompatible strains interact. Vegetative compatibility has been used as a multilocus phenotype in analysis of fungal populations. In some highly clonal populations the vegetative-compatibility phenotype is correlated with pathogenicity. The molecular basis for vegetative compatibility is not well understood. Fourhet loci have been cloned fromNeurospora crasset orPodospora anserina, inch but no two are alike and it is clear that thehet genes themselves do not encode the gene products that are directly responsible for cell death. We suggest that a broader view of vegetative compatibility would include genes that are responsible for prefusion, fusion, and postfusion activities. Postfusion activities could include the fungal apoptotic apparatus since microscopic observations of cell death resemble those in higher plants and animals.     

Regulation of tyrosine biosynthesis in Escherichia coli K-12: isolation and characterization of operator mutants

Mutant strains of Escherichia coli have been isolated in which the synthesis of two of the enzymes involved in tyrosine biosynthesis, 3-deoxy-d-arabinoheptulosonic acid-7 phosphate synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase, is partially constitutive. The mutations involved are closely linked to aroF and tyrA, the structural genes of these enzymes. The gene in which the mutations occur has been designated aroK, and the gene sequence is aroK, aroF, tyrA. In aroK(+)/aroK diploids, the aroK allele only affects the structural genes in the cis position. The mutant allele aroK is not recessive to aroK(+) and aroK/aroK(+) strains exhibit the aroK phenotype of resistance to 4-aminophenylalanine. It is proposed that aroK is an operator locus for an aroF tyrA operon.     

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.     

Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor.

An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition. The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures. A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria. Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene. The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction. The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its six terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues. The E. coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication. Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C. Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C. One of these extragenic suppressors was mapped at 54 min on the E. coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.     

Human alpha-globin gene expression. The dominant role of the alpha 2-locus in mRNA and protein synthesis

The two human alpha-globin genes, alpha 1 and alpha 2, are coexpressed in normal erythroid cells and encode identical alpha-globin protein products. Based upon genetic studies, it has been assumed that these two adjacent and highly homologous genes are equally expressed. In previous studies we have, however, demonstrated that the alpha 2 gene encodes a 2-3-fold higher steady state level of mRNA than the alpha 1 gene. In the present study, we monitor the relative levels of protein production from these two loci by quantitating the synthesis of specific alpha-globin structural mutants encoded by each alpha-globin gene. These values are then used to infer the relative contributions of the normal alpha 1 and alpha 2 loci to total alpha-globin production. The results of eight separate studies, each based upon a different alpha-globin structural mutant mapped to either the alpha 1 or the alpha 2 locus, are internally consistent. The data demonstrate that the alpha 2 gene encodes 2-3-fold more protein than the alpha 1 gene. These results suggest that the human alpha-globin gene cluster contains a major and a minor locus. The dominant expression of the alpha 2 gene predicts a greater impact of mutations at this locus, in comparison to mutations at the alpha 1 locus, in the generation of the alpha-thalassemia phenotype.     

The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper

In Saccharomyces cerevisiae, recessive mutations at the OPI1 locus result in constitutively derepressed expression of inositol 1-phosphate synthase, the product of the INO1 gene. Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high derepressed levels in opi1 mutants. Thus, the OPI1 gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes. In this study, the OPI1 gene was mapped to chromosome VIII and cloned. When transformed into an opi1 mutant, the cloned DNA was capable of complementing the mutant phenotype and restoring correct regulation to the INO1 structural gene. Construction of two opi1 disruption alleles and subsequent genetic analysis of strains bearing these alleles confirmed that the cloned DNA was homologous to the genomic OPI1 locus. Furthermore, the OPI1 gene was found to be nonessential to the organism since mutants bearing the null allele were viable and exhibited a phenotype similar to that of previously isolated opi1 mutants. Similar to other opi1 mutants, the opi1 disruption mutants accumulated INO1 mRNA constitutively to a level 2-3-fold higher than that observed in wild-type cells. The cloned OPI1 gene was sequenced, and translation of the open reading frame predicted a protein composed of 404 amino acid residues with a molecular weight of 40,036. The predicted Opi1 protein contained a well defined heptad repeat of leucine residues that has been observed in other regulatory proteins. In addition, the predicted protein contained polyglutamine residue stretches which have also been reported in yeast genes having regulatory functions. Sequencing of opi1 mutant alleles, isolated after chemical mutagenesis, revealed that several were the result of a chain termination mutation located within the largest polyglutamine residue stretch.     

Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product.

A cloned Neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. Sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic DNA and the normally located gene. Such changes are typical of the duplication-induced premeiotic disruption (the RIP effect) first described by Selker et al. (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987). The mutants had the unusual phenotype of growth on ethanol but not on acetate as the carbon source. In a cross to the wild type of a mutant strain in which the original ectopic gene sequence had been removed by segregation, the acetate nonutilizing phenotype invariably segregated together with a RIP-induced EcoRI site at the normal locus. This mutant was transformed to the ability to use acetate by the cloned sequence. The locus of the mutation, designated acu-8, was mapped between trp-3 and un-15 on linkage group 2. The transcribed portion of the clone, identified by probing with cDNA, was sequenced, and a putative 525-codon open reading frame with two introns was identified. The codon usage was found to be strongly biased in a way typical of most Neurospora genes sequenced so far. The predicted amino acid sequence shows no significant resemblance to anything previously recorded. These results provide a first example of the use of the RIP effect to obtain a mutant phenotype for a gene previously known only as a transcribed wild-type DNA sequence.     

The Arabidopsis root hair cell wall formation mutant lrx1 is suppressed by mutations in the RHM1 gene encoding a UDP-L-rhamnose synthase

Cell and cell wall growth are mutually dependent processes that must be tightly coordinated and controlled. LRR-extensin1 (LRX1) of Arabidopsis thaliana is a potential regulator of cell wall development, consisting of an N-terminal leucine-rich repeat domain and a C-terminal extensin-like domain typical for structural cell wall proteins. LRX1 is expressed in root hairs, and lrx1 mutant plants develop distorted root hairs that often swell, branch, or collapse. The aberrant cell wall structures found in lrx1 mutants point toward a function of LRX1 during the establishment of the extracellular matrix. To identify genes that are involved in an LRX1-dependent developmental pathway, a suppressor screen was performed on the lrx1 mutant, and two independent rol1 (for repressor of lrx1) alleles were isolated. ROL1 is allelic to Rhamnose Biosynthesis1, which codes for a protein involved in the biosynthesis of rhamnose, a major monosaccharide component of pectin. The rol1 mutations modify the pectic polysaccharide rhamnogalacturonan I and, for one allele, rhamnogalacturonan II. Furthermore, the rol1 mutations cause a change in the expression of a number of cell wall-related genes. Thus, the lrx1 mutant phenotype is likely to be suppressed by changes in pectic polysaccharides or other cell wall components.     

Alternative route for biosynthesis of amino sugars in Escherichia coli K-12 mutants by means of a catabolic isomerase.

By inserting a lambda placMu bacteriophage into gene glmS encoding glucosamine 6-phosphate synthetase (GlmS), the key enzyme of amino sugar biosynthesis, a nonreverting mutant of Escherichia coli K-12 that was strictly dependent on exogenous N-acetyl-D-glucosamine or D-glucosamine was generated. Analysis of suppressor mutations rendering the mutant independent of amino sugar supply revealed that the catabolic enzyme D-glucosamine-6-phosphate isomerase (deaminase), encoded by gene nagB of the nag operon, was able to fulfill anabolic functions in amino sugar biosynthesis. The suppressor mutants invariably expressed the isomerase constitutively as a result of mutations in nagR, the locus for the repressor of the nag regulon. Suppression was also possible by transformation of glmS mutants with high-copy-number plasmids expressing the gene nagB. Efficient suppression of the glmS lesion, however, required mutations in a second locus, termed glmX, which has been localized to 26.8 min on the standard E. coli K-12 map. Its possible function in nitrogen or cell wall metabolism is discussed.