Identification of Mom12 and Mom13, two novel modifier loci of ApcMin-mediated intestinal tumorigenesis

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc^Min/+ mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc^Min/+ mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc^Min model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc^Min-mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc^Min/+ mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc^Min/+ controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: (1) Mom12, a dominant modifier linked to the congenic region on chromosome 6 and (2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.Key words: adenomatous polyposis coli, modifier of min, congenic mice, caveolin-1, cancer susceptibility     

Monolayers assembled from a glycolipid biosurfactant from <Emphasis Type="Italic">Pseudozyma</Emphasis> (<Emphasis Type="Italic">Candida</Emphasis>) <Emphasis Type="Italic">antarctica</Emphasis> serve as a high-affinity ligand system for immunoglobulin G and M

A carbohydrate ligand system has been developed which is composed of self-assembled monolayers (SAMs) of mannosylerythritol lipid-A (MEL-A) from Pseudozyma antarctica, serving for human immunoglobulin G and M (HIgG and HIgM). The estimated binding constants from surface plasmon resonance (SPR) measurement were K _a = 9.4 × 10⁶ M⁻¹ for HIgG and 5.4 × 10⁶ M⁻¹ for HIgM, respectively. The binding site was not in the Fc region of immunoglobulin but in the Fab region. Large amounts of HIgG and HIgM bound to MEL-A SAMs were directly observed by atomic force microscopy.     

<Emphasis Type="Italic">KRAS</Emphasis>, <Emphasis Type="Italic">BRAF</Emphasis>, <Emphasis Type="Italic">EGFR</Emphasis> and <Emphasis Type="Italic">HER2</Emphasis> gene status in a Spanish population of colorectal cancer

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical–pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

A novel mouse Chr5 locus <Emphasis Type="Italic">Diht</Emphasis> controls dopamine-induced hypothermia

Mice of the strain C3H.PRI-Flv^r, carrying genetically determined resistance to flaviviruses, have been shown to be more sensitive to the hypothermic effect of dopamine than congenic flavivirus-susceptible C3H/HeJARC mice. In the current study, the greater sensitivity to dopamine-induced hypothermia observed in flavivirus-resistant mice was shown to be dose-dependent, with strain differences being the most prominent at a moderate dose of apomorphine (1 mg/kg). In addition, hypothermic responses to apomorphine were shown to be under developmental regulation; aging increased the potency of apomorphine-induced hypothermia and abrogated strain and sex differences observed in young mice. Linkage analysis of mouse strain-dependent co-inheritance between flavivirus resistance and greater sensitivity to the hypothermic effect of dopamine was performed using two genetically unrelated flavivirus-susceptible and two highly congenic flavivirus-resistant mouse strains in parallel with C3H.PRI-Flv^r-and C3H/HeJARC reference strains. This study has revealed a clear segregation between flavivirus resistance conferred by the Flv locus and sensitivity to dopamine-controlled hypothermia conferred by a novel locus, Diht. Parallel studies in F1 and F2 heterozygote mice showed that the high sensitivity to hypothermic effect of dopamine (Diht^high) is inherited as the Chr5-linked dominant trait. The novel locus, Diht, has been mapped proximal to the Flv locus on a distal part of mouse Chr5 between microsatellite markers D5Mit41 and D5Mit158.     

Identification of Mom12 and Mom13, two novel modifier loci of ApcMin-mediated intestinal tumorigenesis

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc^Min/+ mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc^Min/+ mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc^Min model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc^Min-mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc^Min/+ mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc^Min/+ controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: 1) Mom12, a dominant modifier linked to the congenic region on chromosome 6, and 2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Transfer and expression of <Emphasis Type="Italic">npt</Emphasis>II and <Emphasis Type="Italic">bar</Emphasis> genes in cucumber (<Emphasis Type="Italic">Cucumis satavus</Emphasis> L.)

Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l⁻¹ phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2–6 mg l⁻¹). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l⁻¹) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.     

A single-base deletion mutation in <Emphasis Type="Italic">SlIAA9</Emphasis> gene causes tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) <Emphasis Type="Italic">entire</Emphasis> mutant

The entire (e) locus of tomato (Solanum lycopersicum L.) controls leaf morphology. Dominant E and recessive e allele of the locus produce pinnate compound and complex reduced leaves. Previous research had indicated that SlIAA9, an Aux/IAA gene, was involved in tomato leaf morphology. Down-regulation of SlIAA9 gene by antisense transgenic method decreased the leaf complex of tomato and converted tomato compound leaves to simple leaves. The leaf morphology of these transgenic lines was similar with leaf morphology of tomato entire mutant. In this paper, we report that a single-base deletion mutation in the coding region of SlIAA9 gene results in tomato entire mutant phenotypes.     

Fine-mapping of <Emphasis Type="Italic">qRfg2</Emphasis>, a QTL for resistance to <Emphasis Type="Italic">Gibberella</Emphasis> stalk rot in maize

Stalk rot is one of the most devastating diseases in maize worldwide. In our previous study, two QTLs, a major qRfg1 and a minor qRfg2, were identified in the resistant inbred line ‘1145’ to confer resistance to Gibberella stalk rot. In the present study, we report on fine-mapping of the minor qRfg2 that is located on chromosome 1 and account for ~8.9% of the total phenotypic variation. A total of 22 markers were developed in the qRfg2 region to resolve recombinants. The progeny-test mapping strategy was developed to accurately determine the phenotypes of all recombinants for fine-mapping of the qRfg2 locus. This fine-mapping process was performed from BC₄F₁ to BC₈F₁ generations to narrow down the qRfg2 locus into ~300 kb, flanked by the markers SSRZ319 and CAPSZ459. A predicted gene in the mapped region, coding for an auxin-regulated protein, is believed to be a candidate for qRfg2. The qRfg2 locus could steadily increase the resistance percentage by ~12% across different backcross generations, suggesting its usefulness in enhancing maize resistance against Gibberella stalk rot.     

Increased tolerance to salt stress in the phosphate-accumulating <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants <Emphasis Type="Italic">siz1</Emphasis> and <Emphasis Type="Italic">pho2</Emphasis>

High salinity is an environmental factor that inhibits plant growth and development, leading to large losses in crop yields. We report here that mutations in SIZ1 or PHO2, which cause more accumulation of phosphate compared with the wild type, enhance tolerance to salt stress. The siz1 and pho2 mutations reduce the uptake and accumulation of Na⁺. These mutations are also able to suppress the Na⁺ hypersensitivity of the sos3-1 mutant, and genetic analyses suggest that SIZ1 and SOS3 or PHO2 and SOS3 have an additive effect on the response to salt stress. Furthermore, the siz1 mutation cannot suppress the Li⁺ hypersensitivity of the sos3-1 mutant. These results indicate that the phosphate-accumulating mutants siz1 and pho2 reduce the uptake and accumulation of Na⁺, leading to enhanced salt tolerance, and that, genetically, SIZ1 and PHO2 are likely independent of SOS3-dependent salt signaling.     

Three ENU-induced neurological mutations in the pore loopof sodium channel <Emphasis Type="Italic">Scn8a</Emphasis> (Na<Subscript>v</Subscript>1.6) and a genetically linkedretinal mutation, <Emphasis Type="Italic">rd13</Emphasis>

The goal of The Jackson Laboratory Neuroscience Mutagenesis Facility is to generate mouse models of human neurological disease. We describe three new models obtained from a three-generation screen for recessive mutations. Homozygous mutant mice from lines nmf2 and nmf5 exhibit hind limb paralysis and juvenile lethality. Homozygous nmf58 mice exhibit a less severe movement disorder that includes sustained dystonic postures. The mutations were mapped to the distal region of mouse Chromosome (Chr) 15. Failure to complement a mutant allele of a positional candidate gene, Scn8a, demonstrated that the mutations are new alleles of Scn8a. Missense mutations of evolutionarily conserved residues of the sodium channel were identified in the three lines, with the predicted amino acid substitutions N1370T, I1392F, and L1404H. These residues are located within the pore loop of domain 3 of sodium channel Na_v1.6. The lethal phenotypes suggest that the new alleles encode proteins with partial or complete loss of function. Several human disorders are caused by mutation in the pore loop of domain 3 of paralogous sodium channel genes. Line nmf5 contains a second, independent mutation in the rd13 locus that causes a reduction in cell number in the outer nuclear layer of the retina. rd13 was mapped to the distal 4 Mb of Chr 15. No coding or splice site mutations were detected in Pde1b, a candidate gene for rd13. The generation of three independent Scn8a mutations among 1100 tested G3 families demonstrates that the Scn8a locus is highly susceptible to ENU mutagenesis. The new alleles of Scn8a will be valuable for analysis of sodium channel physiology and disease.(David A. Buchner and Kevin L. Seburn) These authors contributed equally.     

Endopolygalacturonase: a Candidate Gene for <Emphasis Type="Italic">Freestone</Emphasis> and <Emphasis Type="Italic">Melting Flesh</Emphasis>in Peach

Peach fruit are handled, processed, and marketed according to their stone adhesion and fruit softening type. Uncertainty exists over whether these simply inherited traits are controlled by two linked loci, Freestone (F) and Melting flesh (M) or one multi-allelic locus, and whether M is controlled by the cell wall degrading enzyme, endopolygalacturonase. From morphological and molecular analysis of two related segregating populations of peach, we conclude that a single locus containing at least one gene for endopolygalacturonase, controls both F and M with at least three effective alleles. A simple diagnostic PCR test is now available for the three major phenotypes of freestone melting flesh (FMF), clingstone melting flesh (CMF), and clingstone non-melting flesh (CNMF).     

Targeted knockout and <Emphasis Type="Italic">lacZ</Emphasis> reporter expression of the mouse <Emphasis Type="Italic">Tmhs</Emphasis> deafness gene and characterization of the <Emphasis Type="Italic">hscy-2J</Emphasis> mutation

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ^tm1Kjn ) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of β-galactosidase activity in Tmhs ^tm1Kjn heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.     

Ectopic over-expression of two apple <Emphasis Type="Italic">Flowering Locus T</Emphasis> homologues, <Emphasis Type="Italic">MdFT1</Emphasis> and <Emphasis Type="Italic">MdFT2</Emphasis>, reduces juvenile phase in <Emphasis Type="Italic">Arabidopsis</Emphasis>

To get insight into mechanism by which apple tree (Malus domestica Borkh.) regulates flowering, two apple flowering locus T (FT) homologues, MdFT1 and MdFT2, were isolated from the leaf cDNAs of cultivar Gala. The open reading frames (ORFs) of two MdFTs encoded 174 amino acids. The deduced amino acid sequence of MdFT1 and MdFT2 showed 94.3 % similarity to each other, while 72.6 and 76.0 % to AtFT protein, respectively. Semi-quantitative RT-PCR indicated their specific expression in leaves. Visualization of MdFT2-GFP fusion protein demonstrated its localization on membrane. Ectopic overexpression of either MdFT1 or MdFT2 in Arabidopsis significantly induced early flowering by activating the downstream flowering-related genes.