Two genes controlling acute phase responses by the antitumor polysaccharide,lentinan

Lentinan, a -1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) andMus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence lenght polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 andltn1.2) responsible for DT-APR.ltn1.1 is closely linked toD3Mit11 on chromosome 3 andltn1.2 toD11Nds9 on chromosome 11 (P<0.001). The linkage analysis also suggested thatltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that theltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.     

BAC/YAC contigs from the H2-M region of mouse Chr 17 define gene order as Znf173-Tctex5-Mog-D17Tu42-M3-M2

 A yeast artificial chromosome (YAC) contig from the C57BL/6 (H2 ^b ) mouse was created from the major histocompatibility complex (Mhc, H2 in mouse) class Ib subregion, H2-M. It spans approximately 1.2 megabase (Mb) pairs and unites the previous >1.5-Mb YAC contigs (Jones et al. 1995) into a single contig, which includes 21 Mhc class I genes distal to H2-T1. A bacterial artificial chromosome (BAC) contig from the 129 (H2 ^bc ) mouse, spanning approximately 600 kilobases, was also built from Znf173 (Afp, a gene for acid finger protein), through Tctex5 (t-complex testis expressed-5) and Mog (myelin oligodendrocyte glycoprotein), to H2-M2. Twenty-four sequence-tagged site (STS) markers were newly developed, and 35 markers were mapped in the YAC/BAC contigs, which define the marker order as Cen –Znf173–Tctex5 – Mog–D17Tu42–D17Mit232–H2-M3–D17Leh525–H2-M2– Tel. The gene order of Znf173 – Tctex5 – Mog – D17Tu42 is conserved between mouse and human, showing that the middle H2-M region corresponds to the subregion of the human Mhc surrounding HLA-A. Received: 25 July 1997 / Revised: 10 September 1997     

Fine mapping of the human pentraxin gene region on chromosome 1q23

 The 1q21 to 25 region of human chromosome 1 contains genes which encode proteins with immune- and inflammation-associated functions. These include the pentraxin genes, for C-reactive protein (CRP), serum amyloid P (SAP) protein (APCS), and a CRP pseudogene (CRPP1). The region of chromosome 1 containing this cluster is syntenic with distal mouse chromosome 1. We constructed an approximately 1.4 megabase yeast artificial chromosome (YAC) contig with the pentraxin genes at its core. This four-YAC contig includes other genes with immune functions including the FCER1A gene, which encodes the α-subunit of the IgE high-affinity Fc receptor and the IFI-16 gene, an interferon-γ-induced gene. In addition, it contains the histone H3F2 and H4F2 genes and the gene for erythroid α-spectrin (SPTA1). The gene order is cen.-SPTA1-H4F2-H3F2-IFI-16-CRP-CRPP1-APCS-FCER1A- tel. The contig thus consists of a cluster of genes whose products either have immunological importance, bind DNA, or both. Received: 13 December 1995 / 6 February 1996     

Allotetraploidy of Zoysia species with 2n=40 based on a RFLP genetic map

 A RFLP linkage map of Zoysia spp. (2n=40), a warm-season turfgrass, was constructed by using the self-pollinated progenies obtained from an interspecific hybrid. Out of 115 DNA clones tested, 100 (87.0%), including 55 genomic clones, 38 cDNA clones, and seven gene clones encoding photosynthetic enzymes showed allelic-RFLP banding patterns among the parental accessions. We found that 26 probes detected two or more loci segregating in the self-pollinated progenies independently. The RFLP linkage map of Zoysia spp. consists of 115 loci in 22 linkage groups ranging in size from 12.5 cM to 141.3 cM with a total map distance of 1506 cM. Six RFLP loci (5.4%) showed significant segregation distortion (P<0.01). Two loci out of six were mapped to linkage group II, and another two loci were mapped to group VII. In the RFLP linkage map of zoysiagrass, five pairs of linkage groups sharing a series of duplicated loci with approximately the same order were identified. Therefore, we conclude that Zoysia spp. with 2n=40 should be considered as allotetraploids, which might have evolved from progenitors with a basic chromosome number of ten (x=10). Received: 20 March 1998 / Accepted: 17 September 1998     

The human natural killer gene complex is located on chromosome 12p12-p13

 Natural killer (NK) cells preferentially express several type II glycoproteins of the calcium-dependent lectin superfamily. The genes coding for these molecules are clustered on the distal mouse chromosome 6 and on the rat chromosome 4 in a region designated the NK gene complex. To date, no definite evidence of the presence of a NK gene complex has been found in humans. Here we report the assignment by fluorescence in situ hybridization of the CD94 gene to human chromosome 12p12-p13, in the same region where the CD69 and NKG2A genes had been previously mapped. In addition, using a yeast artificial chromosome contig spanning this region we determined that the human CD94, NKG2A, NKG2C, NKG2E, and NKR-P1A (NKR) genes map to the short arm of chromosome 12. The distal to proximal position of these loci are: NKR- CD69 - CD94/NKG2A/NKG2C/NKG2E. These data demonstrate the existence of a human NK gene complex located within a 5.6 cM interval flanked by the genetic markers D12S397 and D12S89. The physical distance spanned by the NK gene complex in humans ranges between 0.7 and 2.4 megabases. Received: 17 January 1997 / Revised: 10 March 1997     

BAC clones and STS markers near the distal breakpoint of the fourth t-inversion, In(17)4d, in the H2-M region on mouse Chromosome 17

The H2-M region is the most distal part of the mouse major histocompatibility complex (Mhc) and is likely to include the distal breakpoint of the fourth t-inversion, In(17)4d. The conserved synteny breakpoint between mouse and human is located in the H2-M region between D17Leh89, a putative olfactory receptor gene, and Pgk2 (phosphoglycerate kinase 2). To analyze the H2-M region, we screened a mouse bacterial artificial chromosome (BAC) library, using the D17Mit64, D17Tu49, D17Leh89, D17Leh467, and Pgk2 markers. Thirty-eight BAC clones were obtained and mapped in five clusters, and 25 sequence-tagged site (STS) markers were newly developed. The regions surrounding D17Tu49 and D17Leh467 are abundant in L1 repeat sequences and may, therefore, be candidates for the breakpoints of conserved synteny and t-inversion. D17Leh89 was linked to D17Mit64 by two contiguous BAC clones. The Aeg1 (acidic epididymal glycoprotein 1) and Aeg2 genes were mapped close to Pgk2, on the same BAC clones. The genetic length between D17Leh89–D17Mit64 and Pgk2–Aeg can be estimated as 0.5–0.7 centiMorgan (cM), and the most distal class I gene, H2-M2, can be placed 0.3–1.0 cM proximal to the t-inversion breakpoint. A recombinational hotspot is suggested to be located between Aeg and Tpx1 in an interspecific cross of (C57BL/6J ×Mus spretus). Received: 23 July 1997 / Accepted: 13 November 1997     

Genetic analysis of the dwarfing gene (Rht8) in wheat. Part I. Molecular mapping of Rht8 on the short arm of chromosome 2D of bread wheat (Triticum aestivum L.)

 Two sets of single chromosome recombinant lines comparing 2D chromosomes from the wheat varieties ‘Ciano 67’ and ‘Mara’ with the common 2D chromosome of ‘Cappelle-Desprez’ in a ‘Cappelle-Desprez’ background were used to detect a diagnostic wheat microsatellite marker for the dwarfing gene Rht8. The genetic linkage maps place the wheat microsatellite marker WMS 261 0.6 cM distal to Rht8 on the short arm of chromosome 2D. By PCR analysis the WMS 261 alleles of ‘Mara’, ‘Cappelle-Desprez’ and ‘Ciano 67’ could be distinguished by different fragment sizes of 192 bp, 174 bp and 165 bp, respectively. A screen of over 100 international varieties of wheat showed that the three allelic variants were all widespread. It also demonstrated that a limited number of varieties carried novel WMS 261 variants of over 200 bp. Following classification of the individual recombinant lines for allelic variants at the WMS 261 locus it was possible to attribute a 7- to 8-cm reduction in plant height with the WMS 261-192-bp allele compared to the WMS 261-174-bp allele in the set of recombinant lines comparing 2D chromosomes of ‘Mara’ and ‘Cappelle-Desprez’. A height reduction of around 3 cm was detected between the WMS 261-174-bp allele and the WMS 261-165-bp allele in the recombinant lines comparing 2D chromosomes of ‘Cappelle-Desprez’ and ‘Ciano 67’. Received: 17 October 1997 / Accepted: 12 November 1997     

Molecular mapping of the photoperiod response gene ea7 in barley

 The gene ea ₇ determining photoperiod insensitivity under short day length was mapped on the short arm of chromosome 6H near the centromere. The gene was linked to the two flanking markers Xmwg2264 and Xmwg916 by 6.7 and 13.0 cM, respectively. Compared to Ppd-H1 (chromosome 2H) and Ppd-H2 (chromosome 1H), ea ₇ determines the strongest effect on flowering time with 55 and 18 days difference compared to photoperiod sensitive genotypes grown under short and long photoperiods, respectively. Allelic and homoeologous relationships to major genes and quantitative trait loci controlling flowering time in barley and wheat are discussed. Received: 10 March 1998 / Accepted: 7 April 1998     

Allelic variants of the human MHC class I chain-related B gene (MICB)

 The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (α1), 3 (α2), 4 (α3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the α2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes. Received: 16 April 1997 / Revised: 19 May 1997     

Localization of the bronx waltzer (bv) deafness gene to mouse Chromosome 5

The bronx waltzer (bv) mutation is an autosomal recessive mutation that is manifested as head tossing and circling in the mouse. The mutation affects the inner hair cells (IHCs) and pillar cells in the organ of Corti of the cochlea and the maculae and cristae of the vestibular part of the inner ear. IHCs begin to degenerate by a controlled mechanism of cell death as early as gestational day 17 (G17) in the basal coil of the cochlea, and few surviving IHCs are seen in the adult. As a first step towards the identification of bv, we analyzed a total of 20 loci in 118 mice from an intraspecific backcross giving the gene order: centromere–D5Mit1–D5Mit73–D5Mit55–[D5Mit12, Nds4 (Afp)]–D5Mit87–[D5Mit205, 20, 88, 208, 93–D5Mit338]–D5Mit25–D5Mit209–bv–D5Mit188–D5Mit367–D5Mit95–D5Mit43–D5Mit102. A total of 701 mice were then analyzed for the markers D5Mit93 and D5Mit95, defining a region of 12.08 cM flanking bv. Mice that were recombinant between D5Mit93 and D5Mit95 were analyzed for D5Mit338, D5Mit25, D5Mit209, bv, D5Mit188, and D5Mit367. bv maps 0.14 cM distal of the marker D5Mit209 and 1.14 cM proximal of the marker D5Mit188 in 701 backcross progeny. Received: 3 March 1997 / Accepted: 30 May 1997     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Regulation of proliferation of UM-SCV-1A and UM-SCV-6 vulvar carcinoma cells by cytokines

 The biology and pathogenesis of vulvar carcinoma are poorly understood at present. In order to understand this disease better, we have used recently developed squamous cell carcinoma lines of the vulva as models. Two cell lines originating from two individuals (UM-SCV-1A and UM-SCV-6) were cultured in vitro in 10% fetal calf serum. The effects of interleukins 10 and 13, interferons α and γ, granulocyte/macrophage-growth-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), and transforming growth factor β (TGFβ) on the proliferation of the cells was investigated by using radioactively labelled uridine as tracer. In addition, an investigation on the molecular structure of extracted cellular DNA was carried out to investigate whether programmed cell death (apoptosis) would be inducible by any of the factors. In UM-SCV-1A cells, interleukin-10 (IL-10) and interleukin-13 (IL-13) caused an approximately 12-fold decrease in DNA synthesis in cells cultured for 72 h (P<0.001), while GM-CSF had no significant effect. TGFβ showed a significant inhibitory effect on deoxyuridine incorporation (P<0.001), which was 2.0- and 4.2-fold at 48 h and 72 h, respectively. TFGα showed a 1.2-fold inhibitory effect on DNA synthesis at 48 h (P<0.01) and a 1.5-fold inhibition at 72 h (P<0.05). Interferon γ (IFNγ) showed an inhibitory effect on DNA synthesis (1.3-fold; P<0.01). In UM-SCV-6 cells, both IL-10 and IL-13 showed inhibitory effects on deoxyuridine incorporation (1.3- and 1.4-fold at 48 h, respectively; P<0.001) that were even more pronounced at 72 h (2.4- and 2.5-fold respectively; P<0.001). IFNγ caused a 3.6-fold inhibition of DNA synthesis by UM-SCV-6 cells at 72 h (P<0.001). Both TFGβ and TNFα inhibited uridine incorporation (3.0- and 1.6-fold at 48 h, respectively; 2.7-fold at 72 h for both factors). GM-CSF inihibited DNA synthesis by UM-SCV-6 cells 1.3- 2.0-fold at 48 h and 72 h, respectively. In dose/response analyses, the effect of INFα on DNA synthesis was inhibitory in both cell lines at 48 h, while stimulatory effects were observed at 72 h. Electrophoretic analyses of DNA isolated from cells cultured in the presence or absence of different factors did not reveal DNA fragmentation. All cytokines, with the exception of IFNα, showed inhibitory effects on DNA synthesis by vulvar carcinoma cells. Of the factors studied, the recently described interleukins 10 and 13 showed potent inhibition of cell growth, encouraging further investigation on the molecular mechanisms of the observed inhibition. Apoptosis does not seem to be induced in the two vulvar carcinoma cell lines by any of the cytokines studied. Received: 26 March 1996 / Accepted: 5 December 1996     

Molecular cloning of the human gene STK10 encoding lymphocyte-oriented kinase, and comparative chromosomal mapping of the human, mouse, and rat homologues

 LOK is a new and unique member of the STE20 family with serine/threonine kinase activity, and its expression is restricted mostly to lymphoid cells in mice. We cloned the cDNA encoding the human homologue of LOK. The amino acid sequence deduced from the cDNA shows a high similarity to that of mouse LOK, with 88% identity as a whole. The kinase domains at the N-terminus and the coiled-coil regions at the C-terminus are particularly conserved, showing 98% and 93% identity, respectively. Western blot analysis with mouse LOK-specific antibody detected 130 000 M _r LOK proteins in human and rat lymphoid cell lines and tissues. The gene encoding the LOK (STK10/Stk10) gene was mapped by fluorescence in situ hybridization to chromosome 5q35.1 in human, chromosome 11A4 in mouse, and chromosome 10q12.3 in rat. By virtue of polymorphic CA repeats found in the 3' untranslated region of the mouse Stk10 gene, the Stk10 locus was further pinpointed to chromosome 11 between D11Mit53 and D11Mit84, using the intersubspecific backcross mapping panel. These results established STK10 as a new marker of human chromosome 5 to define the syntenic boundary of human chromosomes 5 and 16 on mouse chromosome 11. Received: 28 September 1998 / Revised: 2 November 1998     

Mapping of the Gracile Axonal Dystrophy (gad) Gene to a Region between D5Mit197 and D5Mit113 on Proximal Mouse Chromosome 5

The gracile axonal dystrophy (gad) mouse, which shows hereditary sensory ataxia and motor paresis, has been morphologically characterized by the dying back type of axonal degeneration in the nerve terminals of dorsal root ganglion cells and motor neurons. In the present study, using an intraspecific backcross between gad and C57BL/6J mice, the gracile axonal dystrophy (gad) gene was mapped to a region between D5Mit197 and D5Mit113. Estimated distances between gad and D5Mit197 and between gad and D5Mit113 are 0.4 ± 0.3 and 5.0 ± 1.0 cM, respectively. The gene order was defined: centromere- D5Mit81-D5Mit233-D5Mit184/D5Mit254-D5Mit256-D5Mit197-gad-D5Mit113-D5Mit7 . The mouse map location of the gad locus appears to be in a region homologous to human 4p15-p16. Our present data suggest that the nearest flanking marker D5Mit197 provides a useful anchor for the isolation of the gad gene in a yeast artificial chromosome contig.     

Cerebellar deficient folia (cdf): A new mutation on mouse Chromosome 6

Cerebellar deficient folia, cdf, is a spontaneous autosomal recessive mutation in the mouse with unique pathology; the cerebellar cortex of the cdf/cdf mouse has only 7 folia instead of 10, which is the normal count for the C3H/HeJ strain in which this mutation arose. The cerebellum of the cdf/cdf mouse is hypoplastic and contains mineral deposits in the ventral vermis that are not present in controls. We used an intersubspecific intercross between C3H/HeSnJ-cdf/+ and Mus musculus castaneus (CAST/Ei) to map the cdf mutation to Chromosome (Chr) 6. The most likely gene order is D6Mit16–(cdf, D6Mit3)–D6Mit70–D6Mit29–D6Mit32, which positions cdf distal to lurcher (Lc) and proximal to motor neuron degeneration 2 (mnd2). The definitive visible phenotypes and histopathologies of cdf, Lc, and mnd2 support our mapping evidence that cdf is a distinct gene. The novel pathology of cdf should help elucidate the complicated process of cerebellar folia patterning and development. cdf recombined with mouse atonal homolog 1, Math1, the mouse homolog of the Drosophila atonal gene. Received: 2 August 1996 / Accepted: 2 October 1996     

Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

 First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1 _P ) and terminator (PGK1 _T ) sequences on a multicopy episomal plasmid. The resulting construct PGK1 _P -abf B-PGK1 _T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively. Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996     

Construction and characterization of the IGF Arabidopsis BAC library

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10 752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100 kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180 bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80 kb <85% <120 kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing. Received: 26 November 1997 / Accepted: 19 February 1998