Change in the protective activity of the ribosomal fractions of Salmonella typhimurium in mutations in the rfa region and the transduction replacement of the rfb region]

It was shown that mutation in the rfa region causing disturbances in the structure of the basal part of the polysaccharide of the cell wall or the absence of O-specific side chains led to the loss of protective activity of the ribosomal fractions isolated from the cells of the murine typhoid salmonella by sedimentation with dihydrostreptomycine sulphate. Ribosomal fractions isolated from the murine typhoid salmonella transductants with the replaced rfb region failed to protect the animals from the infection with the virulent. S. typhimurium, S. enteritidis cultures. The virulence of the mutants and transductants was also changed in comparison with the initial strains.     

Localization and characterization of the region encoding catabolism of mannopinic acid from the octopine-type Ti plasmid pTi15955

The region of the octopine-type tumor-inducing (Ti) plasmid pTi15955 encoding catabolism of mannopinic acid was localized to an 18-kilobase (kb) segment mapping between Ti plasmid coordinates 70 and 88. A subclone containing only this region normally did not allow utilization of any other mannityl opine. However, spontaneous mutants of the plasmid were isolated that conferred catabolism of agropinic acid. While the mutants remained regulated for mannopinic acid utilization, induction by this opine resulted in increased activity associated with agropinic acid catabolism. Respirometric studies and results from growth assays on mannopinic acid analogues indicated that the genes encoded on the 18-kb subclone were regulated in a wild-type fashion. By means of these analogues, spontaneous constitutive mutants were isolated. In several cases, the mutant phenotype was associated with an insertion event mapping within a 300-base pair region of the subclone insert. Although constitutive for catabolism of mannopinic acid, these mutants remained unable to catabolize any of the other mannityl opines. Segregation studies and genetic reconstitution experiments showed that one of the subclones isolated directly in Agrobacterium was actually composed of two complementing recombinant plasmids contained in the same cell. This indicated that the genes encoding mannopinic acid catabolism were organized into at least two complementation groups. These results point to a high degree of complexity in the organization and regulation of Ti plasmid genes encoding catabolism of a relatively simple carbon source.     

Identification of the pore-forming region of the outer chloroplast envelope protein OEP16

The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids. The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants. Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli. The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein. Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu(73) to Val(91)) did not change the properties of the OEP16 channel. The only exception was a Cys(71)-->Ser mutation, which led to a loss of the CuCl(2) sensitivity of the channel. Analysis of N- and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93.     

Characterization of the rfc region of Shigella flexneri.

The O antigen of the Shigella flexneri lipopolysaccharide (LPS) is an important virulence determinant and immunogen. We have isolated S. flexneri mutants which produce a semi-rough LPS by using an O-antigen-specific phage, Sf6c. Western immunoblotting was used to show that the LPS produced by the semi-rough mutants contained only one O-antigen repeat unit. Thus, the mutants are deficient in production of the O-antigen polymerase and were termed rfc mutants. Complementation experiments were used to locate the rfc adjacent to the rfb genes on plasmid clones previously isolated and containing this region (D. F. Macpherson, R. Morona, D. W. Beger, K.-C. Cheah, and P. A. Manning, Mol. Microbiol 5:1491-1499, 1991). A combination of deletions and subcloning analysis located the rfc gene as spanning a 2-kb region. Insertion of a kanamycin resistance cartridge into a SalI site in this region inactivated the rfc gene. The DNA sequence of the rfc region was determined. An open reading frame spanning the SalI site was identified and encodes a protein with a predicted molecular mass of 43.7 kDa. The predicted protein is highly hydrophobic and showed little sequence homology with any other protein. Comparison of its hydropathy plot with that of other Rfc proteins from Salmonella enterica (typhimurium) and Salmonella enterica (muenchen) revealed that the profiles were similar and that the proteins have 12 or more potential membrane-spanning segments. A comparison of the S. flexneri rfc gene and protein product with other rfc and rfc-like proteins revealed that they have a similarly low percentage of G + C content and have similar codon usage, and all have a high percentage of rare codons. An attempt to identify the S. flexneri Rfc protein was unsuccessful, although proteins encoded upstream and downstream of the rfc gene could be identified. Examination of the distribution of rare or minor codons in the rfc gene revealed that it has several minor codons within the first 25 amino acids. This is in contrast to the upstream gene rfbG, which also has a high percentage of rare codons but whose gene product could be detected. The positioning of the rare codons in the rfc gene may restrict translation and suggests that minor isoaccepting tRNA species may be involved in translational regulation of rfc expression. The low percentage of G + C content of rfc genes may be a consequence of the selection pressure to maintain this form of control.     

Suppression by the DNA fragment of the motX promoter region on long flagellar mutants of Vibrio alginolyticus

The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5 microm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild-type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long-Pof strain V10578. The swimming speed and flagellar length of these transformants were almost equal to the wild-type values. The amounts of PF47 flagellin and PF60 sheath-associated protein, which increased in the long-Pof mutants, were retrieved to almost the wild-type level in the transformants. The plasmid pMF209 contained only a 143 bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long-Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.     

Mutations in the bacteriophage lambda pL/oL region that spontaneously occur in plasmid pRPZ126

In a previous study (Chen and Porter, 1988), we isolated spontaneous mutations in a test plasmid that had occurred under non-selective conditions and assigned them to 1 of 6 different categories or groups. The test plasmid, pRPZ126, is a pBR322 derivative containing the bacteriophage lambda immunity region with the cI857 allele so that plasmid-containing cells shifted to 42 degrees C survive only if the expression of the lambda kil gene is prevented by mutation. 75% of the total spontaneous mutations obtained fall into two of these groups where there is no readily detectable change in plasmid size. The two groups differ in that the plasmids from the group 4 mutations are missing a specific HincII site while the plasmids from the group 5 mutations had no detectable plasmid change whatsoever. In this study, we randomly selected ten group 4 mutants and ten group 5 mutants and sequenced the lambda pL/oL region of the mutant plasmid. Of the ten group 4 mutants (HincII site missing), five involved a 24- or 44-basepair deletion in the pL/oL region of the plasmid. The other five group 4 mutants and four of the ten group 5 mutants were A-T to G-C transitions in the pL/oL region. The remaining six group 5 mutants did not demonstrate any sequence change in the pL/oL region of the plasmids. 8 out of 9 of these transition mutations occurred next to the 3' end of 3 different 5'-PyGGNPuNTG-3' sequences in the lambda operator region, and this same sequence is found adjacent to the A-T to G-C transition hotspot in the lac operator region (Schaaper et al., 1986). The 9th mutation, where the A-T to G-C transition occurred one basepair away from the lambda operator, was adjacent to a very similar sequence.     

New antibiotic resistance Loci in the ribosomal region of yeast mitochondrial DNA

A large number of mitochondrial antibiotic-resistant mutants have been isolated following mutagenesis with manganese. These include several different phenotypic classes of mutants, as distinguished by cross-resistance patterns, that have been found to be allelic at cap1 or ery1; some have been found to be heteroallelic.--Seven chloramphenicol-resistant mutants have been identified that are nonallelic by recombination tests with the three loci (cap1, spi1 and ery1) previously identified in the ribosomal region. Four of these are allelic with each other and define a new locus, cap3; two others are allelic and define another new locus, cap2; the seventh maps at yet a different locus, cap4. One new spiramycin-resistant mutant has been identified that defines still another new locus, spi2. A variety of genetic techniques have been used to map these loci within the ribosomal region of the mitochondrial genome.-Manganese has been shown to be effective in inducing the mutation from omega(-) to omega(n) in many mutants that experience a simultaneous mutation at the closely linked cap1 locus. The omega(n) mutation has also been described in the cap4 mutant, and this locus has been shown to be more closely linked to omega than cap1 is to omega.     

Identification of a sequence containing the positive regulatory region of Saccharomyces cerevisiae gene ENO1

A DNA fragment which contains the 5'-flanking region of the Saccharomyces cerevisiae enolase 1 gene (ENO1) and a portion of the coding sequence was cloned in a plasmid pMC1587. This fragment was fused in frame to the lacZ gene of Escherichia coli. Many mutants which deleted a portion of the 5'-flanking region of ENO1 were isolated from this ENO1-lacZ fusion plasmid by in vitro recombination. Analysis of beta-galactosidase activity of these mutants indicated that the regulatory region responsible for an efficient expression of the ENO1-lacZ fused gene resides within an 86-bp sequence located at -487 to -402 upstream from the start codon of ENO1. We found that the segment encompassing the 86-bp region worked equally well in an inverted orientation, but the tandem duplication of the sequence did not enhance the expression of the fused gene.     

Isolation of transformation-defective, replication-nondefective early region 1B mutants of adenovirus 12.

We isolated three adenovirus 12 early region 1B mutants (in205B, in205C, and dl205) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. These mutants could replicate efficiently in human embryo kidney or KB cells but showed markedly reduced transforming capacities both in vitro and in vivo. In cells infected with the mutants, the early region 1B gene was transcribed efficiently. In cells infected with in205B, the products corresponding to the early region 1B-coded 19,000-molecular-weight polypeptide was detected by in vitro translation but not immunoprecipitated extract of labeled cells. In cells infected with in205C or dl205, the products corresponding to the same polypeptide were not detected by either in vitro translation or immunoprecipitation of labeled cell extracts. The results suggest that the 19,000-molecular-weight polypeptide encoded by early region 1B is required for cell transformation but not for viral propagation.     

A cluster of cell division genes maps to the terC region of the chromosome of Escherichia coli K-12

Thirty-nine cell division mutants were isolated in Escherichia coli K-12 and were mapped in the terminus region of the chromosome, between 33.5 and 36 min. They were obtained by two different approaches involving specific mutagenesis of the terC region. The mutants could be divided into eight classes (I to VIII) based on their map position and phenotype at the restrictive temperature, and constitute a new cell division gene cluster.     

Identification of a small genetic region that encodes orotate phosphoribosyltransferase and orotidylate decarboxylase in Drosophila melanogaster

Summary A variety of tests indicate that a small genetic region in chromosome 3R encodes at least a part of the bienzyme complex containing the last two enzymes of the pyrimidine biosynthetic pathway in Drosophila melanogaster, Complex U. Electrophoretic variants of the last pathway enzyme map to this region and both Complex U activities are proportionally reduced in flies heterozygous for specific deficeencies of the region. Complex U mutants have been isolated that lack both activities. Characteristics of these mutants are discussed relative to other pyrimidine pathway mutants known in Drosophila melanogaster and in mammals.     

Refined genetic analysis of the region II che mutants in Salmonella typhimurium

Summary From a detailed complementation analysis of the region II che mutants of Salmonella typhimurium, we have located five che genes, cheA, cheW, cheR, cheB, and cheY. We have shown that corrections are required in the previous assignment of the mutations in four strains: both SL2514 and SL2515 which have been reported to be cheY mutants are cheR mutants, SL2539 is not a cheA but a cheW mutant, and ST171 which has been reported to be a cheZ mutant is a double mutant with defects in both cheA and cheB. Since ST171 is the only cheZ mutant so far isolated, the idea that the cheZ gene might play an essential role in chemotaxis in S. typhimurium as in Escherichia coli has lost its experimental basis. Furthermore, a number of deletion mutants in region II resulting from the excision of Tn10 have been isolated and analysed. From these experiments, we propose that the gene order in region II is flaK-flaE-motA-motB-cheA-cheW-cheR-cheB-cheY-flaM-flaC, which is identical with that in E. coli.     

Anchorage of the Chinese hamster dihydrofolate reductase gene to the nuclear scaffold occurs in an intragenic region

We have identified a region near the center of the dihydrofolate reductase gene dhfr in Chinese hamster ovary cells that is attached to nuclear scaffolds isolated by extraction with lithium diiodosalicylate. Detailed analysis presented here reveals the presence of only two closely linked sites in 35,000 base-pairs scanned that mediate attachment of the dhfr gene to the nuclear scaffold. Sequence analysis of one of the sites reveals a high A + T content, the presence of cleavage consensus sequences for topoisomerase II, and direct and inverted repeated sequence motifs that are localized to a small region of the attachment site. Attachment of these two regions to the nuclear scaffold is observed in wild-type, hemizygous, and amplified cell lines. Attachment is also retained in dhfr mutants isolated in our laboratory, in which chromosomal lesions have occurred directly adjacent to the scaffold-associated regions. These two regions are not bound to scaffolds prepared from isolated metaphase chromosomes, suggesting that attachment of the dhfr gene is lost during mitosis.     

Characterization of strong polar mutations in a region immediately adjacent to the L-arabinose operator in Escherichia coli B-r

Seven l-arabinose-negative mutations are described that map in three genetically distinct regions immediately adjacent to the araO (operator) region of the l-arabinose operon. All seven mutants revert spontaneously, exhibit a cis-dominant, trans-recessive polarity effect upon the expression of l-arabinose isomerase (gene araA), and fail to respond to amber, ochre, or UGA suppressors. Three of these mutants exhibit absolute polarity and are not reverted by the mutangens 2-aminopurine, diethyl sulfate, and ICR-191. These may have arisen as a consequence of an insertion mutation in gene araB or in the initiator region of the l-arabinose operon. The four remaining mutants exhibit strong but not absolute polarity on gene araA and respond to the mutagens diethyl sulfate and ICR-191. Three of these mutants are suppressible by two independently isolated suppressors that fail to suppress known nonsense codons. Partially polar Ara(+) revertants with lesions linked to ara are obtained from three of the same four mutants. These polar mutants, their external suppressors, and their partially polar revertants are discussed in terms of the mechanism of initiation of expression of the l-arabinose operon.     

A cluster of cell division genes maps to the terC region of the chromosome of Escherichia coli K-12

Thirty-nine cell division mutants were isolated in Escherichia coli K-12 and were mapped in the terminus region of the chromosome, between 33.5 and 36 min. They were obtained by two different approaches involving specific mutagenesis of the terC region. The mutants could be divided into eight classes (I to VIII) based on their map position and phenotype at the restrictive temperature, and constitute a new cell division gene cluster.     

Isolation and characterization of mutants deleted for the sulA-ompA region of the Escherichia coli K-12 chromosome

Abstract We have isolated mutants deleted for different segments of the sulA-ompA region of the Escherichia coli K-12 chromosome using gene fusion techniques. Genetic and physical analysis showed that the deletions ranged from 500 to more than 4000 base pairs (bp) Strains were found in which all, or part, of the sulA and ompA genes had been deleted.     

Control of F plasmid replication by a host gene: evidence for interaction of the mafA gene product of Escherichia coli with the mini-F incC region.

Replication of F (including mini-F) and some related plasmids is known to be specifically inhibited in mafA mutants of Escherichia coli K-12. We have now isolated and characterized mini-F mutants that can overcome the replication inhibition. Such plasmids, designated pom (permissive on maf), were obtained spontaneously or after mutagenesis with hydroxylamine or by transposon (Tn3) insertion. In addition to their ability to replicate in mafA mutant bacteria, the pom mutant plasmids exhibit an increased copy number and resistance to "curing" by acridine dye in the mafA+ host. In agreement with these results, Tn3-induced pom mutants were found to carry Tn3 inserted at the incC region of mini-F DNA, known to be involved in incompatibility, control of copy number, and sensitivity to acridine dye. Furthermore, three of the seven mini-F plasmids tested that carry Tn3 within the tandem repeat sequences of the incC region (previously isolated by other workers) exhibit all the phenotypes of pom plasmids, the ability to replicate in the mafA strain, and high copy number and acridine resistance in the mafA+ strain. The rest of the plasmids that contain Tn3 just outside the tandem repeats remain wild type in all these properties. These results strongly suggest that the putative mafA gene product of host bacteria controls mini-F replication through interaction with the incC region.     

Molecular characterization of the gyrB region of the oral spirochete, Treponema denticola

The nucleotide (nt) sequence of the Treponema denticola (Td) DNA gyrase beta-subunit gene (gyrB) has been determined. Southern blot analysis of Td chromosomal DNA indicated that gyrB is present as a single copy. Approximately 3.2kb of the nt sequence 5' and 0.7kb of nucleotide sequence 3' of gyrB were obtained. Analysis of the deduced amino acid (aa) sequence revealed two complete open reading frames (ORFs) (ORF1 and ORF3) and a truncated ORF (ORF4'). ORF1 has no homology to sequences in the databases, whereas ORF3 and ORF4' have significant homology to several bacterial DnaA (replication initiator) and DnaE (DNA polymerase III) proteins respectively. RT-PCR data showed that orf1-gyrB are co-transcribed, while dnaA-dnaE are co-transcribed but in the opposite direction. These data indicated that the gene organization of the Td gyrB region is unique compared with that of other bacteria. Eighteen putative DnaA boxes with several AT-rich regions were identified in the dnaA-dnaE intergenic region, and three putative DnaA boxes were identified in the gyrB-dnaA intergenic region. Spontaneous coumermycin A(1)-resistant Td mutants were isolated and characterized. The mutants have a >20-fold higher resistance to coumermycin A(1) than wild-type Td. A single point mutation in gyrB that changed GyrB Lys(136) to Glu or Thr appears to be responsible for the coumermycin A(1) resistance.