MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum)

Summary Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed.     

Estimating woody and herbaceous vegetation cover from time series satellite observations

In this paper we test a method to estimate the tree and grass vegetation cover over Australia from satellite-derived normalized difference vegetation index (NDVI) time series (monthly 1981–91, ≈5 km pixels) observations. The evergreen cover is assumed to track along the base of the NDVI time series, which is assumed to be equivalent to the woody vegetation cover. The base of the NDVI time series is estimated using modifications to a classical econometric model (i.e. time series is the sum of trend, seasonal and random components). Estimates of the average evergreen component during 1982–85 and 1986–89 were generally consistent with known vegetation distributions. Changes in evergreen cover were largely restricted to the south-west and south-east of Australia. Those changes were largely the result of differences in rainfall between the two periods. The proposed method for estimating woody vegetation cover is found to be generally robust. However, there are some regions where the grass (or pasture) is mostly evergreen. Some possible refinements are proposed to handle such cases.     

An unusual wheat insertion sequence (WIS1) lies upstream of an alpha-amylase gene in hexaploid wheat, and carries a "minisatellite" array

Summary Comparison of the 5′ flanking regions of three α-amylase genes from chromosome 6B of hexaploid wheat by heteroduplex and sequence analysis revealed the presence of a 1.6 kb stem-loop insertion sequence (WIS1) in one of them. Polymorphism among hexaploid wheat varieties suggests the relatively recent insertion/excision of this sequence from its present position. The complete sequence of the stem-loop insertion shows that it has many of the features found in transposable elements, including target site duplication and terminal inverted repeats. One unusual feature is a tandem array of direct repeats comprising a wheat “minisatellite” sequence. Both the insertion sequence and the minisatellite are found at multiple locations in the wheat genome, but the functional significance of their association in WIS1 is unknown. The minisatellite arrays share a common core structure, and long arrays are polymorphic between different hexaploid varieties.     

Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence

Summary Eight representative recombinant background clones of λEMBL3 were analysed usingKpnI,BamHI,SalI,EcoRI andHindIII digestion. We found that λEMBL3 carries its own left arm in theBamHI cloning site. In this way, recombinant molecules were found to be generated which can grow onEscherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restoredBamHI site at thecos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated,BamHI cloning produces a large palindromic sequence consisting of two polylinkers. ThisBamHI site was incompletely cleaved in all cases analysed. We assume that a part of the λ DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonucleaseBamHI.     

Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm-2a region of chromosome 9 in tomato

Summary A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number of single copy clones and the suitability of these enzymes for the mapping of large DNA fragments was evaluated. Furthermore, five genetically tightly linked single copy clones have been used to begin the construction of a physical map in a region of the genome containing the Tm-2a gene which confers resistance to tobacco mosaic virus. Two of the five clones were found to be on the same 560 kb SalI fragment and therefore are no further apart than that distance. The remaining three markers are distributed over at least 3 million bp, so that the total minimum physical distance of that cluster is at least 4 million bp. The results are discussed with respect to correlations between recombination frequencies and physical distance as well as physical mapping large regions of a complex plant genome like tomato.     

Genomic organization of the ribosomal DNA of sorghum and its close relatives

Summary The structure and organization of the ribosomal DNA (rDNA) of sorghum (Sorghum bicolor) and several closely related grasses were determined by gel blot hybridization to cloned maize rDNA. Monocots of the genus Sorghum (sorghum, shattercane, Sudangrass, and Johnsongrass) and the genus Saccharum (sugarcane species) were observed to organize their rDNA as direct tandem repeats of several thousand rDNA monomer units. For the eight restriction enzymes and 14 cleavage sites examined, no variations were seen within all of the S. bicolor races and other Sorghum species investigated. Sorghum, maize, and sugarcane were observed to have very similar rDNA monomer sizes and restriction maps, befitting their close common ancestry. The restriction site variability seen between these three genera demonstrated that sorghum and sugarcane are more closely related to each other than either is to maize. Variation in rDNA monomer lengths were observed frequently within the Sorghum genus. These size variations were localized to the intergenic spacer region of the rDNA monomer. Unlike many maize inbreds, all inbred Sorghum diploids were found to contain only one rDNA monomer size in an individual plant. These results are discussed in light of the comparative timing, rates, and modes of evolutionary events in Sorghum and other grasses. Spacer size variation was found to provide a highly sensitive assay for the genetic contribution of different S. bicolor races and other Sorghum species to a Sorghum population.     

Clone bank and physical and genetic map of potato chloroplast DNA

Summary Clone banks of PvuII, BamHI and XhoI fragments were generated of the Solanum tuberosum cv Katahdin plastome. These clone banks, in conjunction with molecular hybridization to tobacco ctDNA probes, were used to construct a physical map of potato ctDNA. The potato plastome was found to be a circular molecule of 155–156 Kbp containing two inverted repeat regions of 23–27 Kbp. The arrangement of restriction sites is very similar to that of other Solanaceae plastomes. Heterologous hybridization to known ctDNA encoded gene probes from tobacco allowed us to establish a genetic map of the potato chloroplast genome. The arrangement of these genes on the potato plastome resembles that on most higher plant ctDNAs.     

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Summary Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.Contributions no. 45 and no. 490 from the Kihara Institute for Biological Research, Yokohama City University and the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, respectively.