Trans Complementation Research Articles

The structure of cloned hemolysin DNA from plasmid pHly185

The complete DNA segment coding for hemolysis on Escherichia coli plasmid, pHly185, on to the vector pBR322 has been cloned. The recombinant plasmid, pJS204, coded the production of externalized hemolysin and contained four contiguous EcoRI fragments from the original plasmid. A restriction map of pJS204 was generated with six endonucleases. Deletion mutations were constructed from the recombinant plasmid by excision with either BamHI and PstI and religation. The BamHI deletion removed a 3680-bp portion from the recombinant plasmid and prevented the synthesis of active hemolytic activity. The PstI-generated deletion removed 7800 bp from the opposite end of the Hly sequence and resulted in a plasmid coding for the production of active intracellular hemolytic activity but contained no information for the export of hemolysin. We examined the ability of the deletion mutants of pJS204 to complement each other, and Tn5 insertion mutations in the hly region of the parent plasmid, pHly185. The complementation studies indicated the presence of three separate cistrons able to complement in trans in a recA E. coli host. Two cistrons were found to be required for synthesis and one cistron for export of the hemolysin. The presence of two promoters and the directions of transcription for the synthesis of the hly gene products was also inferred from the complementation studies.

Construction and application of R prime plasmids, carrying different segments of an octopine Ti plasmid from Agrobacterium tumefaciens, for complementation of vir genes

Several R prime plasmids have been obtained with high efficiency, by enclosing the R plasmid replicator, in an R::Ti cointegrate plasmid, between two copies of the transposon Tn1831, in the same orientation. These R primes carry different segments of an octopine Ti plasmid, and are compatible with Ti plasmids. They were used to study genetic complementation of Ti plasmid insertion mutants, outside the T-DNA region, which affected oncogenicity. Complementation was observed in both recombination-proficient and -deficient strains. The complementation in trans indicates that certain functions essential for tumor formation outside the T-DNA region are probably expressed in the bacterium. Therefore, the authors proposed to make a distinction between virulence (Vir) functions and oncogenic (Onc) functions of the octopine Ti plasmid of Agrobacterium tumefaciens. A large R prime was obtained, carrying the whole Ti plasmid, except a 7-Mdalton segment, containing the Ti plasmid replicator region. Strains harboring this plasmid induced normal tumors, showing that the replicator region of the octopine Ti plasmid is dispensible for tumor induction.

Ia specificities on parental and hybrid cells of an I-A mutant mouse strain.

Splenic B cells and B cell blasts from the I-A mutant mouse strain B6.C-H-2bm12 were tested by serology with a series of new monoclonal anti-Iab antibodies. Four out of 5 of those monoclonal antibody-defined specificities that are determined by wild-type I-Ab antigens were undetectable on B6.C-H-2bm12 cells. Specificities both present and absent on mutant cells appear to be determinants on the same wild-type molecule, as indicated by sequential precipitation experiments with soluble H-2b antigens. The lack of expression of certain Ia specificities on mutant cells was found not to be the result of disparate control by the Xid gene, which was previously shown to control the expression of Ia.W39, another specificity absent in B6.C-H-2bm12 mice. Serologic testing of Ia specificities on cells and blasts from F1-hybrid mice suggested that the Iabm12 antigens are codominantly expressed, indicating a failure to detect trans regulation or complementation of the mutant phenotype. Another monoclonal antibody-defined Ia specificity dependent on the expression of the E beta polypeptide was normally expressed in B6.C-H-2bm12 mice. These data thus suggest that the lesion of these mutant mice occurred in the A alpha and/or A beta structural gene, resulting in the loss of several Ia specificities.

Genetic complementation and enzyme correlates at the locus encoding the last two steps of de novo pyrimidine biosynthesis in Drosophila melanogaster.

Eight mutations of the rudimentary-like (r-l) locus were isolated following mutagenesis with ethylmethanesulfonate and inter se crosses revealed three basic complementation groups, using the wing phenotype as an index of complementation. One group consists of three entirely noncomplementing mutants that each specify severe reductions in levels of both r-l-encoded enzymes, orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase). The other two groups consist of complementing mutants, such that any member of one group fully complements all members of the other group. One of these groups consist of two mutants that each specify severely reduced OPRTase, but normal ODCase. The other group consists of three mutants that specify severe OPRTase and ODCase reductions in homoallelic flies, but that appear to contribute OPRTase in certain heteroallelic genotypes. It is concluded that the reciprocal and complementing enzymatic phenotypes of mutants in these two groups account for most instances of genetic trans complementation among r-l mutants. These findings are discussed relative to extant information on OPRTase and ODCase in animals and an hypothesis is developed that the r-l locus encodes a single polypeptide product that contains both enzyme activities.

Biochemical evidence for trans complementation of structural genes in the expression of I-E antigens in F1 hybrids.

Analysis of the tryptic peptides obtained from the Ia.7-positive, "I-E" antigens produced by 4 F1 combinations derived from strain A.TFR5 has provided biochemical confirmation that I-A/I-E gene complementation can occur in the transconfiguration. The data obtained support the concept that structural genes, as opposed to regulatory genes, are the essential elements involved in this complementation and support the assignment of the I-E subregion of strain A.TFR5 as I-Ek. The data support the following assignments of the genes encoding the E beta chains: B10.A(4R) as E beta k, B10.A(5R) as E beta b, and B10.HTT as E beta s. Finally, data are presented demonstrating that the E beta chain derived from the recombinant D2.GD strain partially differs from the E beta d chain derived from a conventional H-2d haplotype strain, B10.D2.

Temperature-sensitive yellow mutants of Chlamydomonas reinhardtii

We have isolated and genetically characterized 10 mutants of Chlamydomonas reinhardtii carrying single, mendelian, temperature-sensitive yellow mutations. The mutants have a yellow phenotype at the restrictive temperature (33°C), but have a wildtype phenotype at the permissive temperature (25°C). Based on complementation and recombination tests, the ten mutations include alleles of two previously described yellow loci (y-1 and y-6) and three new yellow loci (y-8, y-9, and y-10). At the restrictive temperature, y-8, y-9, and y-10 are physiologically similar to other yellow mutants. They accumulate small amounts of protochlorophyllide when grown under dim light, but synthesize normal amounts of chlorophyll when grown in the light. Linkage tests indicate that the three new mutations are not linked to each other. y-8 is linked to y-7 on linkage group III, and y-10 is linked to y-5 and y-6 on linkage group I. y-9 is located on linkage group II. We conclude that the control of light-independent protochlorophyllide reduction is complex, involving several genetic loci which are scattered in the genome and which code for gene products able to complement in trans. Temperature-sensitive alleles at several of the yellow loci suggest that the gene products made by these loci are proteins.

Derivation of a monoclonal antibody that detects an Ia antigen encoded by 2 complementing I-subregions.

A monoclonal antibody designated A303, which reacts with Ia antigens controlled by the complementation of 2 distinct I-subregion genes, is described. Complementation of both I-A and I-EC subregion gene products is necessary for the cytotoxic, direct and 125I-protein A-amplified indirect cell binding, and immunoprecipitation activities of A303. Positive prototype strains of independent origin include H-2k,p, whereas A303-negative haplotypes include H-2b,d,f,r,s,u. Seven recombinant strains, H-2ap5,as1,h4,i3,i5,t3,t4 representing intra I-region cross-over events between positive and negative haplotypes are A303 negative. Trans complementation occurs between the I-Ak allele and the I-EdCd, I-EkCk and I-EkCd alleles, respectively. Analysis of F1 heterozygotes suggests that A303-binding activity is also controlled by gene-dose effects. Thus, the activity of A303 is formally equivalent to a system of coupled complementation previously described for Ir and Is genes.

Dissection of the transposition process: A transposon-encoded site-specific recombination system

Deletions of transposons Tn1 and Tn3 that extend into a region of the transposon that specifies a 19,000 molecular weight protein, are unable to resolve presumptive transposition intermediates in recA strains of Escherichia coli. For example, when transposition of such mutant transposons occurs from replicon A to replicon B, cointegrate molecules containing A and B separated by directly repeated copies of the transposons are efficiently produced. Such cointegrates are stable in a recA strain, but are resolved within a recA+ host into replicons A and B each containing a copy of the transposon. One mutant gives cointegrates that can be complemented to resolve when a wild type Tn3 is present in the same recA cell, whereas another gives cointegrates that cannot be resolved by complementation in trans. We suggest that the first such mutant still carries the sequences necessary for the recombination event whereas the latter has lost them.

Virulent mutants of phage P22: II. Physiological analysis of P22 virB-3 and its component mutations

The virulent mutant of phage P22, virB-3, consists of two mutations: K5, which maps in the c 2 repressor gene, and Vx, which maps between c 2 and c 3. Although neither P22 K5 nor P22 Vx is virulent, each of these mutants can express gene functions not expressed by other nonvirulent phages in the presence of prophage immunity. In mixed superinfection of a lysogen with P22 virB-3, only a small fraction of the yield consists of c +, c 1, or c 2 phage even though the virulent grows normally. This is the phenomenon of replication inhibition. In contrast, P22 K5 and to a lesser extent, P22 Vx show escape from replication inhibition. However, neither P22 K5 nor P22 Vx alone replicates extensively in a lysogen. These mutants complement in trans for phage DNA synthesis and phage production. This result indicates that both P22 K5 and P22 Vx express some of the functions required for phage replication in immune conditions. In addition, P22 K5 kills lysogens at low multiplicities, and lysis is observed when these complexes are treated with chloroform. P22 virB-3 is repressed by the c 2 gene product made by a c + phage in mixed infection of a sensitive host. There is an inverse relationship between the burst size and the multiplicity of infection of the c + phage. P22 virB-3 represses its own growth at high multiplicities even though the K5 mutation maps in the c 2 gene and confers a clear plaque phenotype to phage P22. Introduction of a second c 2 mutation into the P22 virB-3 genome abolishes this multiplicity effect. These findings demonstrate that P22 virB-3 is sensitive to its own repressor and to that of a coinfecting phage bearing a c 2 + allele. We have called this effect multiplicity repression. The residual sensitivity of P22 virB-3 to repressor suggests that at least one of its component mutations is of the operator constitutive type.