TRA Loci Research Articles

Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR.

Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), is of critical importance for immune responses to pathogens and vaccines. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference, allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software. We demonstrate the accuracy of this approach using AIRR-seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) AIRR-seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) AIRR-seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity within the IGH, TRA and TRB loci in different populations and to establish a database of alleles of V and J genes inferred from AIRR-seq data and their population frequencies with free public access through an online database.

Platinum Talen-Mediated Knock-in of Single-Stranded DNA Templates Facilitates Manufacturing of Clinical-Scale Non-Virally Gene-Edited T Cells from Peripheral Blood

Background: During the past decade, technologies that manufacture gene-modified immune cells, such as chimeric antigen receptor T cells, have been rapidly evolved and introduced in human clinics. T cells reprogrammed to express antigen-specific T-cell receptors (TCR-T) are an up-and-coming tool to treat intractable infection or malignant neoplasm in a personalized manner. However, several caveats still exist in the production of TCR-T for clinical use, because competitive surface expression of endogenous and transduced TCRs might hamper desired expression of transduced TCR. Moreover, mispairing of TCR subunits could mediate unexpected harmful immune complications. Therefore, in this study, we aimed to develop a method to introduce any desired TCRs in human primary T cells independent of interference with endogenous TCR by way of targeted knockout of TRA and TRB gene loci using highly-efficient transcription activator-like effector nuclease (TALEN), named Platinum TALEN (Sakuma T, Sci Rep 2013). Methods: We designed and synthesized two pairs of 5'-Cap-1 Platinum TALEN mRNA targeting the gene sequences that encode constant regions of human TRA ( TRAC) and TRB ( TRBC). TCR knockout efficiencies by these TALEN pairs were assessed by flow cytometric analysis using anti-CD3/TCR antibodies. Next, we constructed single-stranded DNA (ssDNA) homology-directed repair templates for the knock-in of desired TCR transgene into the TALEN target sequence of the TRAC locus. As a model TCR, we selected a 1G4 clone that reacts with NY-ESO-1-derived peptide in an HLA-A*02-restricted manner. We delivered these TALEN mRNA pairs and 1G4-encoding ssDNA by electroporation into primary human T cells collected from healthy volunteer adult donors after 72 hours of anti-CD3/CD28 beads activation. Genome-edited T cells were subsequently cultured in Therapeak TM X-VIVO15 (Lonza) supplemented with 10% human AB serum in the presence of 2-mercaptoethanol and interleukin-2 followed by second anti-CD3/CD28 beads activation. Density of T cells were maintained approximately at 1.0-3.0 x10 6/mL and transferred to larger flasks with additional fresh media as appropriate according to cell counts throughout the culture.1G4-transduced T cells were detected and counted by HLA-A*02:01 NY-ESO-1 tetramers at 17-22 days after electroporation. Additionally, we performed optical genome mapping analysis (Bionano Genomics) of Platinum TALEN-treated cells to screen genome-editing-associated large chromosomal structural variations (SVs) including unintended translocation. Results: Platinum TALEN mRNA pairs targeting TRAC and TRBC yielded a high proportion (>70%) of CD3/TCR negative T cells without the cost of compromised cell viability. By co-electroporation of 1G4 ssDNA and these Platinum TALEN mRNA pairs, we successfully obtained an average of 10.5 (range 2.2-19.0) million (n=5)NY-ESO-1-tetramer-positive cells with high viability from 3 millionprimary T cells until 3 weeks after electroporation. By flow cytometric analysis, a proportion of 1G4-tetramer positive cells that express Vβ chains other than Vβ13 (Vβ of 1G4) was approximately 10%. The PD-1-positive fraction in the expanded 1G4-expressing T cells was less than 10%, while levels of TIM-3 expression were 40-60%. These tetramer-positive cells showed specific cytotoxicities against HLA-A*02-positive cells pulsed with cognate NY-ESO-1 peptides. In an optical genome mapping analysis of genomic DNA obtained from TALEN-treated T cells, we found unique SVs with relatively small sizes and low coverage but no large unique SVs with high coverage when compared with control genomic DNA. Additionally, no interchromosomal translocation was observed. Conclusions: We have successfully designed Platinum TALEN mRNA pairs targeting TRAC and TRBC which facilitate the production of genome-edited human primary T cells. We also developed an electroporation and expansion culture protocol that enables us to produce viable genome-edited 1G4-transduced T cells at a large cell dose equivalent to a clinical scale. Collectively, these results suggest that targeted orthotopic knock-in of antigen-specific TCR-encoding ssDNA into the TCR locus by the use of Platinum TALEN is a promising strategy that can be applied for the clinical manufacturing of therapeutic TCR-T cells.

Euroclonality-NGS DNA Capture Panel for Integrated Analysis of IG/TR Rearrangements, Translocations, Copy Number and Sequence Variation in Lymphoproliferative Disorders

Introduction: Current diagnostic standards for lymphoproliferative disorders include detection of clonal immunoglobulin (IG) and/or T cell receptor (TR) rearrangements, translocations, copy number alterations (CNA) and somatic mutations. These analyses frequently require a series of separate tests such as clonality PCR, fluorescence in situ hybridisation and/or immunohistochemistry, MLPA or SNParrays and sequencing. The EuroClonality-NGS DNA capture (EuroClonality-NDC) panel, developed by the EuroClonality-NGS Working Group, was designed to characterise all these alterations by capturing variable, diversity and joining IG and TR genes along with additional clinically relevant genes for CNA and mutation analysis. Methods: Well characterised B and T cell lines (n=14) representing a diverse repertoire of IG/TR rearrangements were used as a proficiency assessment to ensure 7 testing EuroClonality centres achieved optimal sequencing performance using the EuroClonality-NDC optimised and standardised protocol. A set of 56 IG/TR rearrangements across the 14 cell lines were compiled based on detection by Sanger, amplicon-NGS and capture-NGS sequencing technologies. For clinical validation of the NGS panel, clinical samples representing both B and T cell malignancies (n=280), with ≥ 5% tumour infiltration were collected from 10 European laboratories, with 88 (31%) being formalin fixed paraffin-embedded samples. Samples were distributed to the 7 centres for library preparation, hybridisation with the EuroClonality-NDC panel and sequencing on a NextSeq 500, using the EuroClonality-NDC standard protocol. Sequencing data were analysed using a customised version of ARResT/Interrogate, with independent review of the results by 2 centres. All cases exhibiting discordance between the benchmark and capture NGS results were submitted to an internal review committee comprising members of all participating centres. Results: All 7 testing centres detected all 56 rearrangements of the proficiency assessment and continued through to the validation phase. A total of 10/280 (3.5%) samples were removed from the validation analysis due to NGS failures (n=1), tumour infiltration < 5% (n=7), and sample misidentification (n=2). The EuroClonality-NDC panel detected B cell clonality (i.e. detection of at least one clonal rearrangement at IGH, IGK or IGL loci) in 189/197 (96%) B cell malignancies. Seven of the 8 discordant cases were post-germinal centre malignancies exhibiting Ig somatic hypermutation. The EuroClonality-NDC panel detected T cell clonality (i.e. detection of at least one clonal rearrangement at TRA, TRB, TRD or TRG loci) in 70/73 (96%) T cell malignancies. In all 3 discordant cases analysis of benchmark PCR data was not able to detect clonality at any TR loci. Next, we examined whether the EuroClonality-NDC panel could detect clonality at each of the individual loci, resulting in sensitivity values of 95% or higher for all IG/TR loci, with the exception of those where limited benchmark data were available, i.e. IGL (n=3) and TRA (n=7). The specificity of the panel was assessed on benign reactive lesions (n=21) that did not contain clonal IG/TR rearrangements based on BIOMED-2/EuroClonality PCR results; no clonality was observed by EuroClonality-NDC in any of the 21 cases. Limit of detection (LOD) assessment to detect IG/TR rearrangements was performed using cell line blends with each of the 7 centres receiving blended cell lines diluted to 10%, 5.0%, 2.5% and 1.25%. Across all 7 centres the overall detection rate was 100%, 94.1%, 76.5% and 32.4% respectively, giving an overall LOD of 5%. Sufficient data were available in 239 samples for the analysis of translocations. The correct translocation was detected in 137 out of 145 cases, resulting in a sensitivity of 95%. Table 1 shows how translocations identified by the EuroClonality-NDC protocol were restricted to disease subtypes known to harbour those types of translocations. Analysis of CNA and somatic mutations in all samples is underway and will be presented at the meeting. Conclusions: The EuroClonality-NDC panel, with an optimised laboratory protocol and bioinformatics pipeline, detects IG and TR rearrangements and translocations with high sensitivity and specificity with a LOD ≤ 5% and provides a single end-to-end workflow for the simultaneous detection of IG/TR rearrangements, translocations, CNA and sequence variants. Table. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Ferrero:Gilead: Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees; Servier: Speakers Bureau. van den Brand:Gilead: Speakers Bureau. Groenen:Gilead: Speakers Bureau. Brüggemann:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy. Langerak:Gilead: Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; Janssen: Speakers Bureau. Gonzalez:Roche: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.

The T cell receptor (TRA) locus in the rabbit (Oryctolagus cuniculus): Genomic features and consequences for invariant T cells.

The rabbit has been widely used in immunology and infectiology. Rabbit immunoglobulins have been extensively studied, leading to the discovery of their idiotypes, allotypic diversity, and of the diversification of the primary repertoire by hyperconversion. Much less is known about rabbit T cell receptors (TR), especially TRA. This isotype is particularly important for innate-like T cells, which typically express invariant TRA (iTRA). The presence of such cells in the rabbit remains an enigma. Rabbit NKT cells seem to be very rare, and lagomorphs lack MAIT cells. TRAV1, the variable gene expressed in the iTRA of these cells across most mammals, and MR1, the MH1-like receptor that present riboflavin derivatives to MAIT cells, are missing in rabbit. An alternative iTRA has been identified, that may be expressed by new innate-like T cells. To facilitate TRA repertoire analyses in rabbit, we report here a full description of TRA and TRD loci and a subgroup definition based on IMGT® classification. Rabbit TRA rearrangements follow the same temporal pattern that is observed in mouse and human. Rare transcripts expressing TRDV/TRDD/TRDJ rearrangements spliced to TRAC were detected. TRA and TRD genes have been made available in IMGT and IMGT/HighV-QUEST, allowing easy analysis of TRA/TRD RepSeq.

The Florida manatee (Trichechus manatus latirostris) T cell receptor loci exhibit V subgroup synteny and chain-specific evolution

The Florida manatee (Trichechus manatus latirostris) has limited diversity in the immunoglobulin heavy chain. We therefore investigated the antigen receptor loci of the other arm of the adaptive immune system: the T cell receptor. Manatees are the first species from Afrotheria, a basal eutherian superorder, to have an in-depth characterization of all T cell receptor loci. By annotating the genome and expressed transcripts, we found that each chain has distinct features that correlates to their individual functions. The genomic organization also plays a role in modulating sequence conservation between species. There were extensive V subgroup synteny blocks in the TRA and TRB loci between T. m. latirostris and human. Increased genomic locus complexity correlated to increased locus synteny. We also identified evidence for a VHD pseudogene for the first time in a eutherian mammal. These findings emphasize the value of including species within this basal eutherian radiation in comparative studies.

V genes in primates from whole genome sequencing data.

The adaptive immune system uses V genes for antigen recognition. However, the evolutionary diversification and selection processes within and across species and orders remain poorly understood. Here, we studied the amino acid (AA) sequences obtained from the translated in-frame V exons of immunoglobulins (IG) and T cell receptors (TR) from 16 primate species whose genomes have been sequenced. Multi-species comparative analysis supports the hypothesis that V genes in the IG loci undergo birth/death processes, thereby permitting rapid adaptability over evolutionary time. We also show that multiple cladistic groupings exist in the TRA (35 clades) and TRB (25 clades) V gene loci and that each primate species typically contributes at least one V gene to each of these clades. The results demonstrate that IG V genes and TR V genes have quite different evolutionary pathways; multiple duplications can explain the IG loci results, while coevolutionary pressures can explain the phylogenetic results of the TR V gene loci. Our results suggest that there exist evolutionary relationships between V gene clades in the TRA and TRB loci. Due to the long-standing preservation of these clades, such genes may have specific and necessary roles for the viability of a species.

Identification and characterization of TCRγ and TCRδ chains in channel catfish, Ictalurus punctatus.

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) γ and δ were identified by mining of expressed sequence tag databases, and full-length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), diversity (D), joining (J), and constant (C) regions. Three TCRγ V families, seven TCRγ J sequences, and three TCRγ C sequences were identified from sequencing of cDNA. Primer walking on bacterial artificial chromosomes (BACs) confirmed that the TRG locus contained seven TRGJ segments and indicated that the locus consists of (Vγ3-Jγ6-Cγ2)-(Vγ1n-Jγ7-Cγ3)-(Vγ2-Jγ5-Jγ4-Jγ3-Jγ2-Jγ1-Cγ1). In comparison for TCRδ, two V families, four TCRδ D sequences, one TCRδ J sequence, and one TCRδ C sequence were identified by cDNA sequencing. Importantly, the finding that some catfish TCRδ cDNAs contain TCR Vα-D-Jδ rearrangements and some TCRα cDNAs contain Vδ-Jα rearrangements strongly implies that the catfish TRA and TRD loci are linked. Finally, primer walking on BACs and Southern blotting suggest that catfish have four TRDD gene segments and a single TRDJ and TRDC gene. As in most vertebrates, all three reading frames of each of the catfish TRDD segments can be used in functional rearrangements, and more than one TRDD segment can be used in a single rearrangement. As expected, catfish TCRδ CDR3 regions are longer and more diverse than TCRγ CDR3 regions, and as a group they utilize more nucleotide additions and contain more nucleotide deletions than catfish TCRγ rearrangements.

Functional genomics to identify the factors contributing to successful persistence and global spread of an antibiotic resistance plasmid.

BackgroundThe spread of bacterial plasmids is an increasing global problem contributing to the widespread dissemination of antibiotic resistance genes including β-lactamases. Our understanding of the details of the biological mechanisms by which these natural plasmids are able to persist in bacterial populations and are able to establish themselves in new hosts via conjugative transfer is very poor. We recently identified and sequenced a globally successful plasmid, pCT, conferring β-lactam resistance.ResultsHere, we investigated six plasmid encoded factors (tra and pil loci; rci shufflon recombinase, a putative sigma factor, a putative parB partitioning gene and a pndACB toxin-antitoxin system) hypothesised to contribute to the ‘evolutionary success’ of plasmid pCT. Using a functional genomics approach, the role of these loci was investigated by systematically inactivating each region and examining the impact on plasmid persistence, conjugation and bacterial host biology. While the tra locus was found to be essential for all pCT conjugative transfer, the second conjugation (pil) locus was found to increase conjugation frequencies in liquid media to particular bacterial host recipients (determined in part by the rci shufflon recombinase). Inactivation of the pCT pndACB system and parB did not reduce the stability of this plasmid.ConclusionsOur findings suggest the success of pCT may be due to a combination of factors including plasmid stability within a range of bacterial hosts, a lack of a fitness burden and efficient transfer rates to new bacterial hosts rather than the presence of a particular gene or phenotype transferred to the host. The methodology used in our study could be applied to other ‘successful’ globally distributed plasmids to discover the role of currently unknown plasmid backbone genes or to investigate other factors which allow these elements to persist and spread.

On The Organization Of Human T Cell Receptor Loci

T cell repertoire in health and disease is complex, comprised of millions of unique T cell clones defined by hetero-dimeric T cell receptors (TCR). The a and b subunits of the TCR are products of genetic loci consisting of variable (V) and joining (J) segments in the case of TCR a (TRA), and V, J and diversity (D) segments in the case of TCR b (TRB). These gene segments are recombined to yield TCR a (VJ) or TCR b (VDJ). Recently we reported that T cell clonal frequency in normal stem cell donors, from the point of view of TCR b gene segment usage rather than being random, has a fractal ordering (Meier et al, BBMT 2013). Fractals are mathematical constructs characterized by self-similarity at different scales of measurement. Thus whether T cell repertoire is examined from the standpoint of DJ, VDJ or VDJ+nucleotide inserted TCR b clones the frequency distribution remains the same when log transformed data are examined yielding a fractal dimension (FD) of ~ 1.5.In order to identify the origin of the observed fractal ordering of TCR b gene segment containing clones, the distribution of gene segments across the TRA and TRB loci was evaluated for self-similarity. Data describing the numeric positions of the initial and final nucleotides of the start and stop codons respectively, was obtained for each gene segment on the two loci was from the NCBI public data base. Resulting gene segment length and intervening space between successive gene segments (in base pairs) were calculated and log-transformed to assess self-similarity by calculating FD using the formula, FD = Log (magnitude)/Log (scaling factor). Length of individual gene segments was considered as scaling factor and the inter-segment space, as the magnitude of the line segment; FD was calculated for each segment. This demonstrated that the V and J gene segments for both TRA and TRB loci were arranged in a fractal, self-similar manner when the spacing between the loci was considered as a function of the size of the gene segment. Despite the apparent variability in the observed sizes of the V and J gene segments and the spacing between them, on a logarithmic scale there was marked uniformity in their size and spacing, with an average FD of 1.4±0.1 and 1.3±0.2 for TRB, V and J segments respectively (Figure). Corresponding values for TRA locus were 1.5±0.1 and 1.7±0.1, for the V and J segments. [Display omitted] During T cell receptor recombination, the J and the D segments in TRA and TRB loci respectively, are recombined with the V segments therefore the distribution of each V segment in the TRA and TRB loci was determined relative to the position of the J and the D segments in the respective loci, by the using the following formula spacing V-J or V-D = INP-D or J segment / INP nthV segment, (INP: initial nucleotide positions of gene segments). This measure demonstrated a strikingly consistent proportionality across both TRA and TRB loci, and when plotted for successive V segments on the TRB locus, the value declined logarithmically, with a slope of approximately 1.6, and similarly an average slope of 1.3 for the TRA locus, consistent with the earlier observed FD for these loci. We conclude that T cell receptor gene segments are organized in an iterating logarithmically scaled manner. This implies that aside from mechanisms such as the 12/23 rule, gene segment distribution across the T cell receptor loci may help determine the order of recombination, and thus contribute to the observed fractal ordering of the T cell repertoire. Disclosures:No relevant conflicts of interest to declare.

The Magnitude of Thymic Output Is Genetically Determined through Controlled Intrathymic Precursor T Cell Proliferation

The thymus plays a crucial role in providing the immune system with naive T cells showing a diverse TCR repertoire. Whereas the diversity of thymic production is mainly ensured by TCR rearrangement at both the TRA and TRB loci, the number of cells reaching the double-positive differentiation stage defines the extent of thymic output. A quantitative analysis of TCR excision circles (TREC; signal-joint TRECs and DJbetaTRECs) produced at different stages of thymopoiesis was performed in nine laboratory mouse strains. The results clearly demonstrate that the magnitude of thymic output is directly proportional to the extent of proliferation in the double-negative 4 thymocyte subset. Strikingly, intrathymic precursor T cell proliferation was found to be strain dependent, thus suggesting a genetic regulation of thymic output. The inherited character of thymic output was further confirmed by the transmission of the phenotype in a recessive fashion in F(1) progeny of the different parental strains. Our results provide the first demonstration of the genetic regulation of thymic output.

IMGT/GeneInfo: T cell receptor gamma TRG and delta TRD genes in database give access to all TR potential V(D)J recombinations.

BackgroundAdaptative immune repertoire diversity in vertebrate species is generated by recombination of variable (V), diversity (D) and joining (J) genes in the immunoglobulin (IG) loci of B lymphocytes and in the T cell receptor (TR) loci of T lymphocytes. These V-J and V-D-J gene rearrangements at the DNA level involve recombination signal sequences (RSS). Whereas many data exist, they are scattered in non specialized resources with different nomenclatures (eg. flat files) and are difficult to extract.DescriptionIMGT/GeneInfo is an online information system that provides, through a user-friendly interface, exhaustive information resulting from the complex mechanisms of T cell receptor V-J and V-D-J recombinations. T cells comprise two populations which express the αβ and γδ TR, respectively. The first version of the system dealt with the Homo sapiens and Mus musculus TRA and TRB loci whose gene rearrangements allow the synthesis of the αβ TR chains. In this paper, we present the second version of IMGT/GeneInfo where we complete the database for the Homo sapiens and Mus musculus TRG and TRD loci along with the introduction of a quality control procedure for existing and new data. We also include new functionalities to the four loci analysis, giving, to date, a very informative tool which allows to work on V(D)J genes of all TR loci in both human and mouse species. IMGT/GeneInfo provides more than 59,000 rearrangement combinations with a full gene description which is freely available at .ConclusionIMGT/GeneInfo allows all TR information sequences to be in the same spot, and are now available within two computer-mouse clicks. This is useful for biologists and bioinformaticians for the study of T lymphocyte V(D)J gene rearrangements and their applications in immune response analysis.

The TRANSFORMER Genes of the Fern Ceratopteris Simultaneously Promote Meristem and Archegonia Development and Repress Antheridia Development in the Developing Gametophyte

The sex of the haploid gametophyte of the fern Ceratopteris is determined by the presence or absence of the pheromone antheridiogen, which, when present, promotes male development and represses female development of the gametophyte. Several genes involved in sex determination in Ceratopteris have been identified by mutation. In this study, the epistatic interactions among new and previously described sex-determining mutants have been characterized. These results show that sex expression is regulated by two sets of genes defined by the FEM1 and TRA loci. Each promotes the expression of either male or female traits and simultaneosly represses the expression of the other. A model describing how antheridiogen regulates the expression of these genes and the sex of the gametophyte is described. The observation that some gametophytic sex-determining mutants have phenotypic effects on the sporophyte plant indicates that sex determination in the Ceratopteris gametophyte is regulated by a mechanism that also regulates sporophyte development.

Sex and the single cell. I. On the action of major loci affecting sex determination in Drosophila melanogaster.

Sex determination in Drosophila melanogaster is under the control of the X chromosome:autosome ratio and at least four major regulatory genes: transformer (tra), transformer-2 (tra-2), doublesex (dsx) and intersex (ix). Attention is focused here on the roles of these four loci in sex determination. By examining the sexual phenotype of clones of homozygous mutant cells produced by mitotic recombination in flies heterozygous for a given recessive sex-determination mutant, we have shown that the tra, tra-2 and dsx loci determine sex in a cell-autonomous manner. The effect of removing the wild-type allele of each locus (by mitotic recombination) at a number of times during development has been used to determine when the wild-type alleles of the tra, tra-2 and dsx loci have been transcribed sufficiently to support normal sexual development. The wild-type alleles of all three loci are needed into the early pupal period for normal sex determination in the cells that produce the sexually dimorphic (in pigmentation) cuticle of the fifth and sixth dorsal abdominal segments. tra(+) and tra-2(+) cease being needed shortly before the termination of cell division in the abdomen, whereas dsx(+) is required at least until the end of division. By contrast, in the foreleg, the wild-type alleles of tra(+) and tra-2(+) have functioned sufficiently for normal sexual differentiation to occur by about 24 to 48 hours before pupariation, but dsx(+) is required in the foreleg at least until pupariation.--A comparison of the phenotypes produced in mutant/deficiency and homozygous mutant-bearing flies shows that dsx, tra-2 and tra mutants result in a loss of wild-type function and probably represent null alleles at these genes.-All possible homozygous doublemutant combinations of ix, tra-2 and dsx have been constructed and reveal a clear pattern of epistasis: dsx > tra, tra-2 > ix. We conclude that these genes function in a single pathway that determines sex. The data suggest that these mutants are major regulatory loci that control the batteries of genes necessary for the development of many, and perhaps all, secondary sexual characteristics.-The striking similarities between the properties of these loci and those of the homeotic loci that determine segmental and subsegmental specialization during development suggest that the basic mechanisms of regulation are the same in the two situations. The phenotypes and interactions of these sex-determination mutants provide the basis for the model of how the wild-type alleles of these loci act together to effect normal sex determination. Implications of these observations for the function of other homeotic loci are discussed.

Characterization of Pseudomonas aeruginosa derepressed R-plasmids.

A genetic study of conjugal transmissibility of two R-plasmids was undertaken in Pseudomonas aeruginosa. Conjugally derepressed mutants of the R-plasmids were isolated, and examination of 11 independent mutants revealed that 10 were recessive to the wild-type transfer repressor, whereas 1 mutant was cis dominant. Cross-repression was observed between the two R-plasmids, suggesting that they have functionally equivalent systems for regulating the expression of tra loci. The derepressed R-plasmid mutants exhibited several characteristics, in addition to derepressed transfer, that were not expressed by the parental plasmids. These included sensitivity to certain donor-specific phages, inhibition of multiplication of a transducing phage, and, in the one case examined, a high degree of entry exclusion. The coexpression of these different functions suggests that their respective genetic loci are controlled by the same regulatory system as that of tra, or else that they are part of the tra complex.

Genetic loci responsible for incompatibility on a co-integrate plasmid, R100-1.

An R plasmid, R100-1, was mapped previously (Yoshikawa, 1974) by transduction from an integratively suppressed Hfr strain to a recipient with a mutation in gene dnaA. By this method various types of transductants of plasmid R100-1 that exist autonomously or in the integrated state were obtained. Seventy-one such transductants were used in the present study to map gene inc, which is responsible for incompatibility. The results obtained can be explained by either of the following: (i) R100-1 has only a single gene or gene cluster (inc) despite previous work suggesting that this plasmid is a co-integrate of two replicons; (ii) R100-1 possesses more than one inc locus located between the repA and tra loci.

Genetic structure of an R factor conferring ampicillin resistance.

An R factor carrying resistant genes, ampicillin (amp), tetracycline (tet), chloramphenicol (cml), dihydrostreptomycin (sir), and sulfanilamide (sul), was analyzed by three genetic techniques: spontaneous segregation, transduction, and recombination. The R factor was stable in Shigella and Escherichia coli, but in Salmonella the (cml.str.sul) genes were spontaneously lost at a high frequency. Almost all of the transductants with Pl contained the five drug markers, but a very small number of them carried both tet and amp genes, or the (amp.cml.str.sul) genes. In conjugal transfer from a strain carrying a single type of R factor to R+ recipient, the recombination took place between the amp and tet, amp and cml, and between the cml and str genes, at a frequency of 23.5, 39.5, and 3.0%, respectively. Crossover between the amp and transfer (tra) loci was not observed. A circular genetic model is proposed in which the genes are arranged in the order –tet9–tet1-tra.amp–cml–str–.

クックトミート培地に保存中に見られたR<sub>12</sub>(CM・SM・SA)因子の自然消失と非伝達性変異

Escherichia coli K-12 F- harboring R12 (CM. SM. SA) factor was stored in cooked meat medium at room temperature. After 3 years, it was examined for loss of R factor, as well as the genetic properties of this factor. It was found that 90.5% of surviving cells had lost their R factor to be sensitive to chloramphenicol (CM), streptomycin (SM), and sulfanilamide (SA). In the other surviving cells retaining their R factor, no segregation was noticed among resistance markers, So long as examined. Two derivative R factors, R12-5 and R12-22, were proveed to be nontransferable by conjugation, indicating that transferable (tra+) R12 factor had mutated to tra- R factor by storage in cooked meat medium for along time. From the results of a curing experiment and transduction to the rec- strain, it was clarified that the tra- R factors, R12-5 and R12-22, existed extrachromosomally and were replicated (rep+) autonomously in their host. Genetic studies revealed that R12-5 and R12-22 factors were ifm+ (inhibition of F-mating) and irs+ (interference of R factor superinfection), as well as the original R12 factor was. Reversion took place from tra- to tra+ at a frequency of 8.0 to 8.8×10-10, indicating that these were point mutants of the tra loci. The E. coli K-12 strain carrying either tra- R12-5 or tra- R12-22 acquired an ability to transfer its nontransferable R factor by complementation in cooperation with F factor, chromosomal F of the Hfr strain, or with transferable R factor.