Interorganellar gene transfer in bryophytes: the functional nad7 gene is nuclear encoded in Marchantia polymorpha

The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes. Received: 11 March 1997 / Accepted: 20 August 1997     

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

 The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed. Received: 4 April 1997 / Accepted: 30 July 1997     

Genomic structure and chromosomal localization of the mouse Ogg1 gene that is involved in the repair of 8-hydroxyguanine in DNA damage

8-Hydroxyguanine (7,8-dihydro-8-oxoguanine: oh⁸Gua) is a damaged form of guanine induced by oxygen-free radicals and causes GC to TA transversions. Previously we isolated the hOGG1 gene, a human homolog of the yeast OGG1 gene, which encodes a DNA glycosylase and lyase to excise oh⁸Gua in DNA. In this study, we isolated a mouse homolog (Ogg1) of the OGG1 gene, characterized oh⁸Gua-specific DNA glycosylase/AP lyase activities of its product, and determined chromosomal localization and exon-intron organization of this gene. A predicted protein possessed five domains homologous to human and yeast OGG1 proteins. Helix-hairpin-helix and C₂H₂ zinc finger-like DNA-binding motifs found in human and yeast OGG1 proteins were also retained in mouse Ogg1 protein. The properties of a GST fusion protein were identical to human and yeast OGG1 proteins in glycosylase/lyase activities, their substrate specificities, and suppressive activities against the spontaneous mutagenesis of an Escherichia coli mutM mutY double mutant. The mouse Ogg1 gene was mapped to Chromosome (Chr) 6, and consisted of 7 exons approximately 6 kb long. Two DNA-binding motifs were encoded in exons 4 through 5. These data will facilitate the investigation of the OGG1 gene to elucidate the relationship between oxidative DNA damage and carcinogenesis. Received: 17 July 1997 / Accepted: 15 September 1997     

Identification and Isolation of the Indole-3-Pyruvate Decarboxylase Gene from Azospirillum brasilense Sp7: Sequencing and Functional Analysis of the Gene Locus

The root-associated bacterium Azospirillum brasilense Sp7 produces the growth-stimulating phytohormone indole-3-acetic acid (=IAA) via the indole-3-pyruvate pathway. The DNA region containing ipdC, the structural gene for indole-3-pyruvate decarboxylase, was identified in a cosmid gene library of strain Sp7 by hybridization and has been sequenced. Upstream of the gene, two other ORF homologous to gltX and cysS were sequenced that are transcribed in the opposite direction. A functional analysis of the cloned ipdC region has been performed. To test the expression of the gene, a lacZ-Km cartridge was introduced into the gene. By this construct, tryptophan-dependent stimulation of gene expression in A. brasilense Sp7 was observed. Evidences for the existence of another copy of the ipdC gene in the Azospirillum genome are also reported. Received: 31 October 1997 / Accepted: 8 December 1997     

Genetic mapping of the mouse Rab7 gene and pseudogene and of the human RAB7 homolog

Rab proteins are small GTP-ases localized to distinct membrane compartments in eukaryotic cells and regulating specific steps of intracellular vesicular membrane traffic. The Rab7 protein is localized to the late endosomal compartment and controls late steps of endocytosis. We have isolated, by library screening, the 5′ region, including the promoter, of the mouse Rab7 gene and a Rab7 pseudogene. We have mapped, by genetic linkage analysis, the mouse Rab7 gene on Chromosome (Chr) 6 and the Rab7-ps1 pseudogene on Chr 9, where the Rab7 gene has been previously reported to map. By radiation hybrid mapping, we have located the human RAB7 gene on Chr 3, in a region homologous to the mouse Chr 6, where the Rab7 gene maps. Received: 27 October 1997 / Accepted: 1 January 1998     

Marker-assisted dissection of the oligogenic anthracnose resistance in the common bean cultivar, ‘G2333’

 Two independently assorting dominant genes conditioning resistance to bean anthracnose were identified in an F₂ population derived from the highly resistant bean differential cultivar, ‘G 2333’. One gene was allelic to the Co-4 gene in the differential cultivar ‘TO’ and was named Co-4 ² , whereas the second gene was assigned the temporary name Co-7 until a complete characterization with other known resistance genes can be conducted. Two RAPD markers linked to the Co-4 ² allele were identified. One RAPD, OAS13₉₅₀, co-segregated with no recombinants in two segregating populations of 143 F₂ individuals, whereas the second RAPD, OAL9₇₄₀, mapped at 3.9 cM from the Co-4 ² allele. Two 24-mer SCAR primers (SAS13), developed from the OAS13₉₅₀ RAPD marker, were dominant and polymorphic, similar to the original RAPD, and supported the tight linkage between the marker(s) and the Co-4 ² allele. The markers were present in germplasm with known resistance alleles at the Co-4 locus. The presence of the markers in two other differential cultivars not previously characterized and in four navy bean cultivars suggests the existence of a gene family for anthracnose resistance at or near the Co-4 locus. Since the Co-7 gene was present only in germplasm which also possessed the Co-4 ² and Co-5 genes, the SAS13 markers were used in combination with standard inoculation techniques to identify F₃ lines in which the Co-7 gene was homozygous and the Co-4 ² allele was absent. A similar strategy of marker-assisted dissection is proposed to identify resistant lines in which the Co-5 gene is absent and the Co-7 gene is present by selecting against the OAB3₄₅₀ marker, which has been shown previously to be linked to the Co-5 gene. These genes cannot be distinguished using traditional screening methods since all current races of the pathogen virulent to the Co-5 gene are avirulent to the Co-4 ² and Co-7 genes. We describe the use of molecular markers tightly linked to resistance genes to facilitate the identification of an uncharacterized resistance gene for which no discriminating race of the pathogen is known. Received: 22 March 1997 / Accepted: 15 July 1997     

Molecular examination of a chromosome region that controls resistance to potato Y and A potyviruses in potato

 The gene Ry _adg that confers resistance to potato Y potyvirus (PVY) in the cultivated potato [Solanum tuberosum subsp. andigena, line 2x(v-2)7] is located on chromosome XI in a segment that contains three other known resistance genes in other syntenic solanaceous species. One of them is the gene N that controls resistance to tobacco mosaic tobamovirus in tobacco and has previously been isolated and sequenced. Three sequence-related, resistance gene-like (RGL) DNA fragments (354–369 bp) highly homologous to the gene N were PCR-amplified from the potato line 2x(v-2)7. Two RGL fragments (79 and 81% homologous to the N gene) co-segregated with Ry _adg among the 77 F1 progeny tested. These RGLs may originate from a resistance gene family on chromosome XI. The potato line 2x(v-2)7 also expressed resistance to potato A potyvirus (PVA), which was controlled by another locus on chromosome XI mapped ca. 6.8 cM distal to Ry _adg . Received: 18 December 1997 / Accepted: 30 December 1997     

Specific induction of secondary product formation in transgenic plant cell cultures using an inducible promoter

This paper describes the artificial induction of secondary metabolite production in transgenic plant cell cultures using a recombinant, inducible plant promoter. The bacterial gene ubiC from Escherichia coli encodes the enzyme chorismate pyruvate lyase (CPL) which catalyses the conversion of chorismate to 4-hydroxybenzoate (4HB). This gene was fused to the tetracycline-inducible plant promoter Triple-Op. After transformation into Nicotiana tabacum W38 TET, transgenic cell cultures were established. Addition of chlorotetracycline to the medium led to specific induction of CPL activity. The optimal chlorotetracycline concentration was approximately 2 mg/l medium. Three to 5 h after induction, the ubiC mRNA concentration reached a maximum, while highest specific CPL activity was detected after 8 days. The artificial secondary metabolite 4HB was converted to glucosides, and their accumulation reached maximum levels after 5 weeks of subculture. The induction was reversible. Received: 31 May 1997 / Revision received: 22 August 1997 / Accepted: 30 September 1997     

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

A serine-to-proline mutation in the copper-transporting P-type ATPase gene of the macular mouse

We have investigated the cDNA sequence of the copper-transporting P-type ATPase (Atp7a) gene of the macular mouse, a model for human Menkes disease. A point mutation (T to C) that results in substitution of proline for serine in a putative eighth transmembrane domain of the ATP7A was identified. This contrasts with abnormalities identified in the Atp7a of other mottled mouse strains: lack of expression of Atp7a mRNA in the dappled mouse, and a splicing mutation in the blotchy mouse. Received: 1 December 1996 / Accepted: 20 January 1997     

The mouse homolog of FRG1, a candidate gene for FSHD, maps proximal to the myodystrophy mutation on chromosome 8

The human autosomal dominant neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) is associated with deletions within a complex tandem DNA repeat (D4Z4) on Chromosome (Chr) 4q35. The molecular mechanism underlying this association of FSHD with DNA rearrangements is unknown, and, thus far, no gene has been identified within the repeat. We isolated a gene mapping 100 kb proximal to D4Z4 (FSHD Region Gene 1:FRG1), but were unable to detect any alterations in total or allele-specific mRNA levels of FRG1 in FSHD patients. Human Chr 4q35 exhibits synteny homology with the region of mouse Chr 8 containing the gene for the myodystrophy mutation (myd), a possible mouse homolog of FSHD. We report the cloning of the mouse gene (Frg1) and show that it maps to mouse Chr 8. Using a cross segregating the myd mutation and the European Collaborative Interspecific Backcross, we showed that Frg1 maps proximal to the myd locus and to the Clc3 and Ant1 genes. Received: 24 September 1996 / Accepted: 7 February 1997     

Cytogenetic rearrangements involving the loss of the Sonic Hedgehog gene at 7q36 cause holoprosencephaly

Holoprosencephaly (HPE) is a genetically heterogeneous disorder that affects the midline development of the forebrain and midface in humans. As a step toward identifying one of the HPE genes, we have set out to refine the HPE3 critical region on human chromosome 7q36 by analyzing 34 cell lines from families with cytogenetic abnormalities involving 7q, 24 of which are associated with HPE. Genomic clones surrounding the DNA marker D7S104, which has previously been shown to be in the HPE3 critical region, have been examined by fluorescent in situ hybridization and microsatellite analysis of our panel of patient cell lines. We report the analysis of a cluster of four translocation breakpoints within a 300-kb region of 7q36 that serves to define the minimal critical region for HPE3 and that has directed the search for candidate genes. The human Sonic Hedgehog (hSHH) gene maps to this region and has been shown to be HPE3 on the basis of mutations within the coding region of the gene. We present evidence that cytogenetic deletions and/or rearrangements of this region of chromosome 7q containing Sonic Hedgehog, and translocations that may suppress Sonic Hedgehog gene expression through a position effect are common mechanisms leading to HPE. Received: 23 December 1996 / Accepted: 17 March 1997     

Evolutionary and polymorphic organization of the knotted1 homeobox in cereals

 Homeobox genes are the master control genes harbouring the homeobox which is crucial for developmental associated functions. One homeobox gene, knotted1, which has a role in leaf development, is conserved in plants and might have arisen from a single ancestral gene. Using PCR, we identified multiple kn1 homeoboxes in diverse cereals and showed a cereal/ species-specific organization correlating them to evolutionary changes. We postulate the insertion of a large intron preceded by duplication of the kn1 homeobox in the lineage leading to rice. Received: 17 October 1997 / Accepted: 9 December 1997     

IRS-PCR-based genetic mapping of the huntingtin interacting protein gene (HIP1) on mouse Chromosome 5

Huntington's disease (HD) is a devastating central nervous system disorder. Even though the gene responsible has been positionally cloned recently, its etiology has remained largely unclear. To investigate potential disease mechanisms, we conducted a search for binding partners of the HD-protein huntingtin. With the yeast two-hybrid system, one such interacting factor, the huntingtin interacting protein-1 (HIP-1), was identified (Wanker et al. 1997; Kalchman et al. 1997) and the human gene mapped to 7q11.2. In this paper we demonstrate the localization of the HIP1 mouse homologue (Hip1) into a previously identified region of human-mouse synteny on distal mouse Chromosome (Chr) 5, both employing an IRS-PCR-based mapping strategy and traditional fluorescent in situ hybridization (FISH) mapping. Received: 7 August 1997 / Accepted: 5 September 1997     

Ammonium repression of the nitrite-nitrate ( nasAB ) assimilatory operon of Azotobacter vinelandii is enhanced in mutants expressing the nifO gene at high levels

A number of Tn5 mutants were isolated which were unable to fix nitrogen and showed enhanced ammonium repression of the nitrate/nitrite assimilation genes. They also had reduced nitrate reductase activity under fully inducing conditions. Insertions were localized within the nifB gene, and inability to fix nitrogen was shown to be due to disruption of the nifB gene. However, enhanced ammonium repression proved to be the result of constitutive expression of the downstream nifO gene from an `out' promoter present in Tn5. Our results suggest that molybdenum metabolism might function as a regulatory factor that acts through the nitrate reductase. Received: 4 December 1996 / Accepted: 27 March 1997