Duplication of the MHC-linked Xenopus complement factor B gene

We have previously reported the molecular cloning of the mammalian major histocompatibility complex (MHC) class III gene, complement factor B (Bf) from Xenopus laevis, and linkage of the gene to the frog MHC. Here, we estimated the copy number of the Xenopus Bf gene by genomic Southern blotting analysis and demonstrated that Xenopus laevis has two copies of the Bf gene. Both genes co-segregated with the MHC-linked HSP70 genes among 19 offspring of an f/r × f/r cross, indicating a close linkage of the two Bf genes to the frog MHC. Both genes are transcribed and contain open reading frames. When compared with the previously determined cDNA sequence (Xenopus Bf A), the predicted amino acid sequence of the second cDNA species (Xenopus Bf B) shows 82% overall identity. Polymerase chain reaction analysis indicated that all of the partially inbred frogs with the f, r, g, and j MHC haplotypes, as well as 12 outbred frogs tested have both Bf genes, suggesting that the duplicated Bf genes are stable genetic traits in Xenopus laevis.     

Dynamics of localization and protein composition of plastid nucleoids in light-grown pea seedlings

Summary Localization and protein composition of plastid nucleoids was analyzed in light-grown pea seedlings at various stages of leaf development. In young plastids of unopened leaf buds, nucleoids were abundant and localized in the periphery of plastids, whereas, in mature leaves, chloroplasts contained nucleoids within narrow spaces restricted by thylakoids or grana. The migration of nucleoids into the interior of plastids preceded the formation of grana, and hence, the maturation of the photosynthetic apparatus. The protein composition of nucleoids was considerably different in young plastids and mature chloroplasts. Polypeptides with a molecular mass of 70–100 kDa predominated in the nucleoids of young plastids, whereas polypeptides with molecular mass of 20–30 kDa were abundant in the nucleoids of mature chloroplasts. Immuno-blot analysis with antibodies against the nucleoids of young plastids identified various polypeptides that were significantly more abundant in the nucleoids of young plastids than in the nucleoids of mature chloroplasts. These results demonstrate that plastid nucleoids are subject to dynamic changes in both localization and composition during the normal development of chloroplasts in the light.Abbreviations DAPI 4,6-diamidino-2-phenylindol - DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Saturation mapping with subclones of YACs: DNA marker production targeting the rice blast disease resistance gene, Pi-b

Saturation mapping of a very small genomic region is indispensable for map-based cloning. We applied a method based on sub-cloning and the Southern-hybridization technique for generating RFLP markers directly from yeast artificial chromosomes (YACs). Two YACs overlapping each other and covering the locus of the rice blast resistance gene, Pi-b, were used to construct a plasmid sub-library. Rice-specific and single-copy clones suitable as probes for RFLP analysis were selected from this sub-library by hybridization to the blots of digested DNAs of rice, YACs, and yeast. As a result, 22 markers were produced within a small chromosomal region including Pi-b. This case study shows that overlapping YACs known to cover the gene of interest are very useful in fine-scale physical mapping leading to map-based cloning of the target gene. Received: 2 May 1996 / Accepted: 2 August 1996     

Ordered YAC Clone Contigs Assigned to Rice Chromosomes 3 and 11

Yeast artificial chromosome (YAC) clones were arranged on thepositions of restriction fragment length polymorphism (RFLP)and sequence-tagged site (STS) markers already mapped on thehigh-resolution genetic maps of rice chromosomes 3 and 11. Froma total of 416 and 242 YAC clones selected by colony/Southernhybridization and polymerase chain reaction (PCR) analysis,238 and 135 YAC clones were located on chromosomes 3 and 11,respectively. For chromosomes 3 and 11, 24 YAC contigs and islandswith total coverage of about 46% and 12 contigs and islandswith coverage of about 40%, respectively, were assigned. Althoughmany DNA fragments of multiple copy marker sequences could notbe mapped to their original locations on the genetic map bySouthern hybridization because of a lack of RFLP, the physicalmapping of YAC clones could often assign specific locationsof such multiple copy sequences on the genome. The informationprovided here on contig formation and similar sequence distributionrevealed by ordering YAC clones will help to unravel the genomeorganization of rice as well as being useful in isolation ofgenes by map-based cloning.     

High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper

We report here an improved protocol for the preparation of full-lengthcDNA libraries that improves the previously reported method(Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics,137, 327–336), that allows long cDNAs to be cloned moreefficiently. One potential disadvantage of the original biotinylatedCAP trapper protocol is the exposure of mRNA to chemical andenzymatic attacks during the biotinylation of the cap structure,before the first-strand cDNA synthesis (and selection of full-lengthcDNA by biotinylated cap). Here, we show that the biotinylationof the cap structure is very specific and effective even ifbiotinylation is performed on the mRNA/cDNA hybrid producedby the first-strand cDNA synthesis reaction. Consequently, mRNAremains protected from chemical and enzymatic degradation duringthe overnight biotinylation step, thus making it possible toselect full-length cDNAs of longer average size. We herein reportthe efficiency and specificity of the new version of the protocolfor cap structure biotinylation and capture of full-length cDNA.     

Construction of YAC Contigs on Rice Chromosome 5

A physical map of rice chromosome 5 was constructed with yeastartificial chromosome (YAC) clones along a high-resolution molecularlinkage map carrying 118 DNA markers distributed over 123.7cM of genomic DNA. YAC clones have been identified by colonyand Southern hybridization for 105 restriction fragment lengthpolymorphism (RFLP) markers and by polymerase chain reaction(PCR) screening for 8 sequence-tagged site (STS) markers and5 randomly amplified polymorphic DNA (RAPD) markers. Of 458YACs, 235 individual YACs with an average insert length of 350kb were selected and ordered on chromosome 5 from the YAC library.Forty-eight contigs covering nearly 21 Mb were formed on thechromosome 5; the longest one was 6 cM and covered 1.5 Mb. Thelength covered with YAC clones corresponded to 62% of the totallength of chromosome 5. There were many multicopy sequencesof expressed genes on chromosome 5. The distribution of manycopies of these expressed gene sequences was determined by YACSouthern hybridization and is discussed. A physical map withthese characteristics provides a powerful tool for elucidationof genome structure and extraction of useful genetic informationin rice.     

Nucleotide Sequence of a 28-kb Mouse Genomic Region Comprising the Imprinted Igf2 Gene

The mouse insulin-like growth factor II gene (Igf2) is physicallylinked to the insulin II gene (Ins2) and both are subject totissue-specific genomic imprinting. The paternal-specific expressionof Igf2 has been associated with hypermethylation of some CpGsites in the 5' flanking region and in the body of the gene.As a first step in analyzing the structural features of thisimprinted locus, we here report the complete nucleotide sequenceof Igf2, including all introns and the intergenic region adjacentto Ins2. This 28-kb segment of mouse chromosome 7 exhibits 80%overall identity with the corresponding rat sequence and hasa high GC content of 52%. In addition to the known CpG islandwithin the second Igf2 promoter, another island was identifiedapproximately 2 kb 5' to the first exon. Other features of thislocus include a 35-fold tandem repeat of an 11-bp sequence thatoverlaps Igf2 pseudo-exon 2, and a B2 repeat element in theintergenic region between Ins2 and Igf2. The GC-richness andthe presence of CpG islands associated with tandem repeats arecommon features of imprinted genes and thus may play a rolein the imprinting mechanism.     

Rice molecular genetic map using RFLPs and its applications

In the past decade, notable progress has been made in rice molecular genetic mapping using genomic or cDNA clones. A total of over 3000 DNA markers, mainly with RFLPs, have been mapped on the rice genome. In addition, many studies related to tagging of genes of interest, gene isolation by map-based cloning and comparative mapping between cereal genomes have advanced along with the development of a high-density molecular genetic map. Thus rice is considered a pivotal plant among cereal crops and, in addition to Arabidopsis, is a model plant in genome analysis. In this article, the current status of the construction of rice molecular genetic maps and their applications are reviewed.     

In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of 6 heterocyclic amines, Trp-P-1 (25 mg/kg), Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg) and PhIP (40 mg/kg), in mouse liver, lung, kidney, brain, spleen, bone marrow and stomach mucosa. Mice were sacrificed 1, 3, and 24 h after intraperitoneal injection. Trp-P-2, IQ, MeIQ, and MeIQx yielded statistically significant DNA damage in the stomach, liver, kidney, lung and brain; Trp-P-1 in the stomach, liver and lung; and PhIP in the liver, kidney and brain. None of the heterocyclic amines induced DNA damage in the spleen and bone marrow. Our results suggest that the alkaline SCG assay applied to multiple organs is a good way to detect organ-specific genotoxicity of heterocyclic amines in mammals.