Molecular analysis of the rat MHC. II. Isolation of genes that map to the RT1.E-grc region

An initial mapping analysis of growth and reproduction complex (grc) and grc+ genomic DNA identified several restriction fragment length polymorphisms specific for the grc region of the MHC. To analyze further the genomic organization and structure of the grc, a cosmid library was constructed from a grc+-bearing strain (R21). One cosmid cluster, encompassing 41.4 kb of DNA, contained four, or possibly five, class I genes that mapped to the RT1.E-grc region Two unique non-class I fragments were isolated from certain cosmids within this cluster. These fragments were hybridized to genomic DNA derived from five rat strains (BIL/2, R18, R21, R22, and BIL/1), and the results showed that grc-bearing rats have a deletion of at least 3.1 kb of DNA in the region immediately adjacent to the MHC. The loss of the genes in this region is probably the cause of the growth and reproductive defects in these animals and probably also of their increased susceptibility to chemical carcinogens.     

Physical mapping of theE/C andgrc regions of the rat major histocompatibility complex

 Alignment of class I-hybridizing cosmids from an R21 (A ^l B ^l D ^l E ^u grc ⁺ ) genomic DNA library gave two contigs: one [150 kilobases (kb)] encompassed the E/C region, or a large part thereof, and the other (110 kb) contained the grc region which has genes influencing resistance to chemical carcinogens (rcc), fertility (ft), and growth (dw-3). Amplification of gene sequences in the four cosmids in the E/C region using E ^u -specific and LW2 (RT1.C)-specific primers showed that each cosmid contained both E ^u -like and C-like genes. They are clearly different but closely associated, and they show some variation from the prototypic E (E ^u ) and C (LW2) genes, respectively. Comparison of DNA from grc ⁺ and grc ^– strains of rats showed that the deletion in the grc ^– strains was approximately 50 kb, and that it was located on two of the three cosmids in the grc-region contig. The use of specific class I probes showed that the grc region contained tandemly duplicated RT1.O-RT1.N genes and that the RT.BM1 loci lay outside of the grc region. Neither contig reacted with probes specific for class II, TNFA, Hsp70, or RT1.M genes. The data presented here and the previous data in the literature (summarized in Gill et al. 1995) suggest that the gene order in the major histocompatibility complex (MHC) and MHC-linked region of the rat is: A-E/C-grc-M. Received: 6 November 1995 / Revised: 24 January 1996     

Histopathology and cell culture characteristics of liver cells from grc − and grc + rats given diethylnitrosamine

The histopathological response and cell culture characteristics of liver cells from the R16 (grc ^–) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/ kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats. The DEN exposure induced hydropicl vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc rats. Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry. Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gammaglutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth. Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage.Abbreviations DEN diethylnitrosamine - grc growth and reproduction complex - GGT gamma-glutamyl transpeptidase - MHC major histocompatibility complex     

Role of IS1 in the conversion of virulence (Vi) antigen expression in Enterobacteriaceae

Summary When Escherichia coli HB101 harbors pWR127, a plasmid comprising the viaB gene from Citrobacter freundii WR7004 and the ColEl-derived pACKC1, the strain produces the virulence (Vi) antigen. Vi antigen expression is abolished (Vi^– phenotype), however, when an IS1 or IS1-like DNA element inserts into the viaB region. To determine the sites of IS1 insertion, pWR127 DNAs extracted from 95 independently isolated Vi^– strains were analyzed by digestion with the restriction endonuclease PstI and agarose gel electrophoresis. Ten insertion sites were found distributed nonrandomly in an area of about 1.3 kb. Nine Vi⁺ strains (two Citrobacter, two E. coli, and five Salmonella strains), four of which contain pWR127, were then tested for the presence of IS1 by DNA-DNA hybridization. Of the nine strains, five were stable Vi⁺ and did not contain IS1. The other four which generated Vi^– strains, contained IS1. When pRR134, a plasmid that contains IS1 was transferred into a stable Vi⁺ Salmonella typhimurium strain carrying pWR127 (OU5140), Vi^– strains were produced from which pWR127 derivatives carrying IS1 inserts could be isolated. It appears, therefore, that the presence of an IS1I or IS1-like element in a strain is required for conversion of the Vi⁺ expression state to the Vi^– expression state.     

Conservation of the central MHC genome: PFGE mapping and RFLP analysis of complement,HSP70, and TNF genes in the goat

A degree of conservation of the genes located between class II and class I [central major histocompatibility complex (MHC) genes] is apparent among mammalian species including primates and the mouse. Few others have been analyzed. The caprine MHC is of particular interest, since it has recently been observed that susceptibility to a lentivirus-induced polyarthritis (caprine arthritis) segregates with serologically defined MHC class I antigens. This arthritis resembles, in a number of respects, rheumatoid arthritis in man. Human cDNA probes were used to examine the caprine central MHC and class I and II genes by restriction fragment length polymorphism (RFLP) and by pulsed field gel electrophoresis (PFGE) in order to define the polymorphism and linkage of central MHC genes to class I and class II genes. An outbred population of dairy goats (Saanen, British Alpine, Anglo Nubian, and Toggenberg) was examined for class I and class II RFLPs. Both regions were found to be highly polymorphic. The number of fragments hybridizing to an HLA-B7 probe after Eco RI, Bam HI, Bgl II, or Hind III digestion suggests there may be 10–13 class I genes. The degree of polymorphism was comparable to that reported in the mouse. Limited polymorphism was found in the central MHC genes. The caprine C4 and CYP21 genes were duplicated and demonstrated RFLP with Bam HI, Hind III, Eco RV, and Taq I. An infrequent Taq I C2 polymorphism was found. PFGE revealed substantial conservation of both the order and linkage of the central MHC genes when compared with mous and man. C4, C2, CYP21, HSP70, and tumor necrosis factor (TNF) genes are all located within 800 kilobase (kb) of the class I loci. Distant from the class I region, the C4, C2, and CYP21 genes are linked on a short genomic segment (180 kb Not I and 190 kb Pvu I fragments). HSP70 cohybridizes with the complement genes on a 380 kb Mlu I fragment. Linkage of HSP70, TNF, and class I genes was found on a single Not I fragment (610 kb). TNF and class I cohybridize on Pvu I (730 kb) and Not I (610 kb) fragments. Conservation of a similar central MHC genomic structure across species argues for functional interaction between the central MHC genes. We postulate selection for these central MHC genes through their role as non antigen-specific regulators of immune response.     

Nuclear tRNATyr genes are highly amplified at a single chromosomal site in the genome of Arabidopsis thaliana

Summary We have examined the organization of tRNA^Tyr genes in three ecotypes of Arabidopsis thaliana, a plant with an extremely small genome of 7 × 107 bp. Three tRNA^Tyr gene-containing EcoRI fragments of 1.5 kb and four fragments of 0.6, 1.7, 2.5 and 3.7 kb were cloned from A. thaliana cv. Columbia (Col-O) DNA and sequenced. All EcoRl fragments except those of 0.6 and 2.5 kb comprise an identical arrangement of two tRNA^Tyr genes flanked by a tRNA^Ser gene. The three tRNA genes have the same polarity and are separated by 250 and 370 bp, respectively. The tRNA^Tyr genes encode the known cytoplasmic tRNA_GA ^Tyr. Both genes contain a 12 by long intervening sequence. Densitometric evaluation of the genomic blot reveals the presence of at least 20 copies, including a few multimers, of the 1.5 kb fragment in Col-O DNA, indicating a multiple amplification of this unit. Southern blots of EcoRl-digested DNA from the other two ecotypes, cv. Landsberg (La-O) and cv. Niederzenz (Nd-O) also show 1.5 kb units as the major hybridizing bands. Several lines of evidence support the idea of a strict tandem arrangement of this 1.5 kb unit: (i) Sequence analysis of the EcoRI inserts of 2.5 and 0.6 kb reveals the loss of an EcoRI site between 1.5 kb units and the introduction of a new EcoRI site in a 1.5 kb dimer. (ii) Complete digestion of Col-O DNA with restriction enzymes which cleave only once within the 1.5 kb unit also produces predominantly 1.5 kb fragments. (iii) Partial digestion with EcoRI shows that the 1.5 kb fragments indeed arise from the regular spacing of the restriction sites. The high degree of sequence homology among the 1.5 kb units, ranging from 92% to 99%, suggests that the tRNA^Ser/tRNA^Tyr cluster evolved 1–5 million years ago, after the Brassicaceae diverged from the other flowering plants about 5–10 million years ago.     

Location of antibiotic resistance determinants, copy control, and replication functions on the double-selective streptococcal cloning vector pGB301

Summary The physical map of the streptococcal cloning vector pGB301 (9.8 kb) has been extended by localizing the cleavage sites of restriction endonucleases AvaII, AvaI, BclI, BstEII, and PvuII. The latter four enzymes cleaved pGB301 at unique sites. Insertion of chromosomal DNA from Staphylococcus aureus strain 3Ar^–m^– into the single BstEII site of pGB301 led to inactivation of the plasmid's chloramphenicol resistance determinant. Twelve deletion derivatives of pGB301 were isolated either by in vitro manipulation of pGB301 or as spontaneous deletion mutants following transformation of Challis by mixtures of recombinant plasmids. The overlapping deletions which spanned a continuous sequence of 7.7 kb ranged in size from 0.3 kb to 4.1 kb and allowed to localize the chloramphenicol and MLS-resistance determinants, the copy control function, and the replication region on the physical map of pGB301. Plasmid pGB301 together with its deletion mutants constitutes a valuable tool for further molecular cloning experiments in streptococci.     

Identification of an antigenic peptide derived from the cancer-testis antigen NY-ESO-1 binding to a broad range of HLA-DR subtypes

NY-ESO-1 is a SEREX-defined cancer-testis antigen of which several MHC I, but only few MHC II–restricted epitopes have been identified. Searching for highly promiscuous MHC II–restricted peptides that might be suitable as a CD4⁺ stimulating vaccine for many patients, we used the SYFPEITHI algorithm and identified an NY-ESO-1–derived pentadecamer epitope (p134–148) that induced specific CD4⁺ T-cell responses restricted to the HLA-DRB1 subtypes *0101, *0301, *0401, and *0701 that have a cumulative prevalence of 40% in the Caucasian population. The DR restriction of the p134–148 pentadecamer was demonstrated by inhibition with an HLA-DR antibody and a functional peptide displacement titration assay with the pan-DR-binding T-helper epitope PADRE as the competitor. The natural processing and presentation of this epitope was demonstrated by recognition of an NY-ESO-1⁺ melanoma cell line by T cells with specificity for p134–148. The pentadecamer p134–148 was able to induce CD4⁺ responses in 4/38 cancer patients tested. However, no strict correlation was found between CD4⁺ T-cell responses against p134–148 reactivity and anti-NY-ESO-1 antibody titers in the serum of patients, suggesting that CD4⁺ and B-cell responses are regulated independently. In conclusion, p134–148 holds promise as a broadly applicable peptide vaccine for patients with NY-ESO-1–positive neoplasms.Abbreviations °C degree Celsius - CD cluster of differentiation - CTA cancer-testis antigen - DC dendritic cells - Gy gray - mM millimolar - ng nanogram - PADRE pan-DR-binding T-helper epitope - pp65 phosphoprotein 65 - SSP-PCR sequence-specific primer PCR - v/v volume per volume This study was supported by BIOMED II (CT BMH4-C98-3589) of the European Commission, Pf-135/7-1, and by Kompetenznetz Maligne Lymphome (TP 11) of the BMBF.     

Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid

The parental strain (A⁺T⁺) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A⁺T^–), catalase A (A^–T⁺) or both catalases (A^–T^–), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two oleic acid-grown A⁺-strains (A⁺T⁺ and A⁺T^–) high catalase activities were found; catalase activity invariably remained low in the A^–T⁺ strain and was never detected in the A^–T^– strain. The levels of -oxidation enzymes in oleic acid-grown cells of the parental and all mutant strains were not significantly different. However, cytochrome C peroxidase activity had increased 8-fold in oleic acid grown A^– strains (A^–T⁺ and A^–T^–) compared to parental strain cells. The degree of peroxisomal proliferation was comparable among the different strains. Catalase A was shown to be located in peroxisomes. Catalase T is most probably cytosolic in nature and/or present in the periplasmic space.     

Characterization of a hemin-storage locus ofYersinia pestis

Summary The pigmentation phenotype (Pgm⁺) ofYersinia pestis refers to temperature-dependent storage of hemin as well as expression of a number of other physiological characteristics. Spontaneous mutation to a Pgm^– phenotype occurs via a large chromosomal deletion event and results in the inability to express the Pgm⁺ characteristics. In this study, we have used transposon insertion mutants to define two regions of a hemin-storage (hms) locus. A clone (pHMSI) encompassing this locus reinstates expression of hemin storage (Hms⁺) inY. pestis spontaneous Pgm^– strains KIM and Kuma but not inEscherichia coli. Complementation analysis using subclones of pHMS1 inY. pestis transposon mutants indicates that both regions (hmsA andhmsB), which are separated by about 4 kb of intervening DNA, are essential for expression of the Hms⁺ phenotype. The 9.1-kb insert of pHMS1 contains structural genes encoding 90-kDa, 72-kDa, and 37-kDa polypeptides. Two-dimensional gel electrophoresis analysis of cells from Pgm⁺, spontaneous Pgm^–, and Hms^– transposon strains, as well as a spontaneous Pgm^– strain transformed with pHMS1, indicated that two families of surface-exposed polypeptides (of about 87 and 69-73 kDa) are associated with the Hms⁺ phenotype.     

Characterization of the class III region in different MHC haplotypes by pulsed-field gel electrophoresis

The class III region of the human major histocompatibility complex (MHC) in sevenHLA haplotypes has been analyzed using pulsed-field gel electrophoresis (PFGE), restriction enzymes that cut genomic DNA infrequently, and Southern blotting. In particular, extensive mapping with the enzymeBss HII, which generates fragments in the size range 8–280 kilobases (kb), has revealed that in the haplotypes studied the DNA content of the class III region does not appear to vary other than as previously observed at theC4 andCYP21 loci.     

Loss ofHindIII cleavage sites in thed-amino acid oxidase gene in some inbred strains of mice

Summary d-Amino acid oxidase cDNA was amplified by a polymerase chain reaction using RNA extracted from the mouse kidney. When digested withHindIII, the cDNAs of the BALB/c and ddY/DAO^– mice were cleaved into two fragments whereas the cDNA of the ddY/DAO⁺ mice was not. Sequencing revealed that nucleotide-471 of the cDNAs was G in the BALB/c and ddY/DAO^– mice whereas it was substituted for C in the ddY/DAO⁺ mice. This base substitution was the cause of the failure of the cleavage of the cDNA of the ddY/DAO⁺ mice.Examination of other strains of inbred mice showed thatd-amino-acid oxidase cDNAs of A/J, AKR, C57BL/6, CD-1, CF#1, ICR, DBA/2, NZB and NZW mice were cleaved withHindIII into two fragments whereas those of C3H/He, CBA/J and NC mice were not. Genomic DNAs extracted from the mice of these 15 strains were digested withHindIII and hybridized withd-amino-acid oxidase cDNA. A 18.2-kb fragment hybridized with the probe in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice whereas two fragments of 12 kb and 6.2 kb hybridized in the other mice. These results are consistent with those of the cDNAs, confirming the loss of theHindIII cleavage site in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice. The Southern hybridization revealed a loss of a differentHindIII cleavage site in the A/J, AKR, C57BL/6, DBA/2, ICR and NZB mice.The substitution at nucleotide-471 should cause a substitution of an amino acid residue. However, this substitution did not affect catalytic activity ofd-amino acid oxidase.     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.     

High-Resolution Genetic Map of the Human Glutamyl Aminopeptidase Gene (ENPEP)

The murine B-lymphocyte differentiation antigen BP-1/6C3 has been identified as glutamyl aminopeptidase (EAP), the gene symbol for which isENPEP.Using genomic DNA encoding for human EAP as a probe, we identified theENPEPgene location on human chromosome 4q25 by polymerase chain reaction analysis of a human/rodent somatic cell hybrid mapping panel and by fluorescencein situhybridization. Using a radiation hybrid panel, the gene order aroundENPEPwas determined to be centromere–D4S1236–(570 kb)–ENPEP–(210 kb)–D4S262–(270 kb)–D4S953–(270 kb)–D4S474–(570 kb)–IF. The linkage ofENPEPto complement factor I (IF) confirms the human chromosome band 4q25 localization predicted from the chromosomal location of murineENPEP.HumanENPEPthus provides an additional marker for the long arm of chromosome 4 that should facilitate studies of this genomic region.     

The feline major histocompatibility complex is rearranged by an inversion with a breakpoint in the distal class I region

In order to determine the genomic organization of the major histocompatibility complex (MHC) of the domestic cat (Felis catus), DNA probes for 61 markers were designed from human MHC reference sequences and used to construct feline MHC BAC contig map spanning ARE1 in the class II region to the olfactory receptor complex in the extended class I region. Selected BAC clones were then used to identify feline-specific probes for the three regions of the mammalian MHC (class II–class III–class I) for radiation hybrid mapping and fluorescent in situ hybridization to refine the organization of the domestic cat MHC. The results not only confirmed that the p-arm of domestic cat B2 is inverted relative to human Chromosome 6, but also demonstrated that one inversion breakpoint localized to the distal segment of the MHC class I between TRIM39 and TRIM26. The inversion thus disjoined the ~2.85 Mb of MHC containing class II–class III–class I (proximal region) from the ~0.50 Mb of MHC class I/extended class I region, such that TRIM39 is adjacent to the Chromosome B2 centromere and TRIM26 is adjacent to the B2 telomere in the domestic cat.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

Regulatory sequences of the Sgs-4 gene of Drosophila melanogaster analysed by P element-mediated transformation

The X chromosomally located allele Sgs-4 ^c for a larval secretion protein of Drosophila melanogaster is normally expressed in female larvae of the strain Oregon R and is hyperexpressed in male larvae exhibiting dosage compensation; the allele Sgs-4 ^d in the strain Samarkand is weakly expressed and is not hyperexpressed in male larvae showing a dosage effect. P element-mediated transformation of upstream DNA sequences from both alleles combined with Sgs-4 ^d coding and downstream sequences was performed to localize sequences which are responsible for the level of gene expression and for hyperexpression of Sgs-4 ^c in male larvae. Our results demonstrate that weak expression and dosage effect are inherited with the upstream region from –1 to –838. This Samarkand fragment differs from the homologous Oregon R region only by a C to T transiion at –344 which lies within an assumed binding sequence for the ecdysone receptor complex of dyad base symmetry. Replacing the Samarkand upstream region from –1 to –838 by the Oregon R region restores normal Sgs-4 expression and dosage compensation. Hyperexpression in male larvae displays high sensitivity to position effect and is nearly completely inhibited in one transformed line under heterozygous conditions. The integration of an Sgs-4 ^d transposon into a weak spot of polytene chromosome 2L results in a decrease in gene expression. The GTT- and GT-rich regions at –1.2 and –2.0 kb do not obviously influence Sgs-4 expression but possibly play a role in induction of stage-specific chromosome puffing.     

Alloreactive T-cell clones. Ly phenotypes predict both function and specificity for major histocompatibility complex products

We have studied the association of Ly phenotype with function and specificity for major histocompatibility complex (MHC) products by examining the properties of 21 T-cell clones derived from B10 anti-B 10.D2 and B10.A anti-B10.D2 mixed lymphocyte cultures (MLC). T cells were selected after MLC solely on the basis of Ly phenotype, cloned by limiting dilution, and tested for stability of Ly phenotype, function and specificity for class I or class II MHC products. Sixteen Ly-1⁺2^– and five Ly-1^–2⁺ T-cell clones were tested. The clones selected for the Ly-1 ⁺2^– phenotype maintained this phenotype, expressed helper but not lytic function, and recognized class II MHC products (I-A^d or I-Ed). All Ly-1^–2^– clones maintained this phenotype, possessed cytolytic but not helper activity, and recognized class I MHC products (D^d and L^d). Our data therefore confirm at the clonal level the original observations of a remarkably consistent correlation between Ly markers, MHC specificity, and. function. They suggest that the expression of Ly antigens on T-cell clones forms part of a genetic program for each of these specialized cells that also determines their function and MHC specificity.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - TCGF T cell growth factor (Interleukin-2) - Con A Concanavalin A - DME Dulbecco's modified Eagle's medium - PHA phytohaemagglutinin - LPS lipopolysaccharide - TRF-C T cell replacing factor required for induction of cytolytic cells from thymocytes - PBS phosphate-buffered saline (pH 7.4)     

Identification of a genomic region that complements a temperature-sensitive, high CO2-requiring mutant of the cyanobacterium, Synechococcus sp. PCC7942

Summary In a temperature-sensitive, high CO₂-requiring mutant of Synechococcus sp. PCC7942, the ability to fix intracellularly accumulated inorganic carbon was severely impaired at non-permissive temperature (41° C). In contrast, inorganic carbon uptake and ribulose-1,5-bisphosphate carboxylase activity in the mutant were comparable to the respective values obtained with the wild-type strain. The mutant was transformed to the wild-type phenotype (ability to form colonies at non-permissive temperature under ordinary air) with the genomic DNA of the wild-type strain. A clone containing a 36 kb genomic DNA fragment of the wild-type strain complemented the mutant phenotype. The complementing activity region was associated with internal 17 kb SmaI, 15 kb HindIII, 3.8 kb BamHI and 0.87 kb Pstl fragments. These 4 fragments overlapped only in a 0.4 kb HindIII-PstI region. In the transformants obtained with total genomic DNA or a plasmid containing the 3.8 kb BamHI fragment, the ability to fix intracellular inorganic carbon was restored. Southern hybridization and partial nucleotide sequence analysis indicated that the cloned genomic region was located approximately 20 kb downstream from the structural genes for subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. The cloned region was transcribed into a 0.5 kb mRNA. These results indicate that the cloned genomic region of Synechococcus sp. PCC7942 is involved in the efficient utilization of intracellular inorganic carbon for photosynthesis.