Population heterogeneity of the Hpa I restriction site associated with the beta globin gene: implications for prenatal diagnosis

The Hpa I restriction endonuclease site polymorphism that results in some human beta globin genes being contained in a 13-kilobase (kb) DNA restriction fragment rather than in the usual 7.6-kb fragment has been reported to be in linkage disequilibrium with the beta S mutation. The frequency of the 13-kb fragment among Baltimore black sickle cell (SS) disease patients (58%) is lower than that reported for San Francisco black SS disease patients (87%) and similar to that reported for such New York patients (59%). There is, then, considerable heterogeneity among American black populations. Therefore, for the purposes of prenatal diagnosis, the frequency in the particular population at risk should be established. When the frequency of association of the 13-kb fragment and the beta S mutation is low, the linkage phase must also be established. When the linkage phase is known, the Hpa I pattern alone can exclude SS disease 54% of the time for Baltimore AS X AS couples.     

Identification of a beta-thalassemia mutation associated with a novel haplotype of RFLPs.

The study of the molecular defects that result in beta-thalassemia in Mediterraneans has uncovered a large number of unique mutations. This information is already being utilized for prenatal diagnosis of pregnancies at risk. Here, we report the definitive identification, by molecular cloning, of the beta-thalassemia mutation associated with a Mediterranean chromosome bearing a novel haplotype of restriction fragment length polymorphisms (RFLPs) in the beta gene cluster that has been previously designated as haplotype X. The thalassemia mutation was identified as a T----C base substitution at IVS-1 position 6, a mutation previously described in association with haplotype VI. We describe the use of the restriction enzyme SfaNI for the detection of this mutation and point out a possible pitfall that should be avoided if such an approach is used for the detection of this mutation, which appears to be a common cause of mild beta+-thalassemia in some populations.     

Ribosomal genes of the loach Misgurnus fossilis L. Isolation and restriction analysis

The ribosomal DNA of the teleost fish--loach has been isolated from sperm DNA by CsCl density gradient centrifugation. The rDNA sediments on density gradients by two heavy satellites beta = 1.715 and greater than 1.720. The DNA of the first satellite (1.715) was separated and treated by restrictases EcoRI and BamHI. It was shown that there are two EcoRI-sites in rDNA of loach, locating in 18S and 28S rRNA coding sequences. From tandem of repeating ribosomal genes EcoRI cuts out the fragment with homogeneous length-3 megadaltons (constant fragment) and heterogeneous population of fragments 11-13 megadaltons (major) and 7-8 megadaltons (minor fraction). The constant fragment contains mostly 28S coding sequence, and the heterogeneous fragment--18S coding sequence. The data indicate that the ribosomal genes of the loach as well as other higher eucaryotes were organized in genome as tandem of repeating units with heterogeneous length (10-16 megadaltons, 14.5-24 kb) due to heterogeneity of the length of nontranscribed spacer.     

The extent of linkage disequilibrium in four populations with distinct demographic histories

The design and feasibility of whole-genome-association studies are critically dependent on the extent of linkage disequilibrium (LD) between markers. Although there has been extensive theoretical discussion of this, few empirical data exist. The authors have determined the extent of LD among 38 biallelic markers with minor allele frequencies >.1, since these are most comparable to the common disease-susceptibility polymorphisms that association studies aim to detect. The markers come from three chromosomal regions-1,335 kb on chromosome 13q12-13, 380 kb on chromosome 19q13.2, and 120 kb on chromosome 22q13.3-which have been extensively mapped. These markers were examined in approximately 1,600 individuals from four populations, all of European origin but with different demographic histories; Afrikaners, Ashkenazim, Finns, and East Anglian British. There are few differences, either in allele frequencies or in LD, among the populations studied. A similar inverse relationship was found between LD and distance in each genomic region and in each population. Mean D' is.68 for marker pairs <5 kb apart and is.24 for pairs separated by 10-20 kb, and the level of LD is not different from that seen in unlinked marker pairs separated by >500 kb. However, only 50% of marker pairs at distances <5 kb display sufficient LD (delta>.3) to be useful in association studies. Results of the present study, if representative of the whole genome, suggest that a whole-genome scan searching for common disease-susceptibility alleles would require markers spaced < or = 5 kb apart.     

The age of human mutation: genealogical and linkage disequilibrium analysis of the CLN5 mutation in the Finnish population.

Variant late infantile neuronal ceroid lipofuscinosis (vLINCL) is an autosomal recessive progressive encephalopathy of childhood enriched in the western part of Finland, with a local incidence of 1 in 1500. We recently assigned the locus for vLINCL, CLN5, to 13q21.1-q32. In the present study, the haplotype analysis of Finnish CLN5 chromosomes provides evidence that one single mutation causes vLINCL in the Finnish population. Eight microsatellite markers closely linked to the CLN5 gene on chromosome 13q were analyzed, to study identity by descent by shared haplotype analysis. One single haplotype formed by flanking markers D13S160 and D13S162 in strong linkage disequilibrium (P < .0001) was present in 81% of disease-bearing chromosomes. Allele 4 at the marker locus D13S162 was detected in 94% of disease-bearing chromosomes. To evaluate the age of the CLN5 mutation by virtue of its restricted geographical distribution, church records were used to identify the common ancestors for 18 vLINCL families diagnosed in Finland. The pedigrees of the vLINCL ancestors merged on many occasions, which also supports a single founder mutation that obviously happened 20 to 30 generations ago (i.e., approximately 500 years ago) in this isolated population. Linkage disequilibrium was detected with seven markers covering an extended genetic distance of 11 cM, which further supports the young age of the CLN5 mutation. When the results of genealogical and linkage disequilibrium studies were combined, the CLN5 gene was predicted to lie approximately 200 - 400 kb (total range 30 - 1360 kb) from the closest marker D13S162.     

A Mutant Brassica napus (Canola) Population for the Identification of New Genetic Diversity via TILLING and Next Generation Sequencing

We have generated a Brassica napus (canola) population of 3,158 EMS-mutagenised lines and used TILLING to demonstrate that the population has a high enough mutation density that it will be useful for identification of mutations in genes of interest in this important crop species. TILLING is a reverse genetics technique that has been successfully used in many plant and animal species. Classical TILLING involves the generation of a mutagenised population, followed by screening of DNA samples using a mismatch-specific endonuclease that cleaves only those PCR products that carry a mutation. Polyacrylamide gel detection is then used to visualise the mutations in any gene of interest. We have used this TILLING technique to identify 432 unique mutations in 26 different genes in B. napus (canola cv. DH12075). This reflects a mutation density ranging from 1/56 kb to 1/308 kb (depending on the locus) with an average of 1/109 kb. We have also successfully verified the utility of next generation sequencing technology as a powerful approach for the identification of rare mutations in a population of plants, even in polyploid species such as B. napus. Most of the mutants we have identified are publically available.     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Batten disease gene, CLN3: linkage disequilibrium mapping in the Finnish population, and analysis of European haplotypes.

The gene for Batten disease (juvenile-onset neuronal ceroid lipofuscinosis, or Spielmeyer-Sjögren disease), CLN3, maps to 16p11.2-12.1. Four microsatellite markers--D16S288, D16S299, D16S298, and SPN--are in strong linkage disequilibrium with CLN3 in 142 families from 16 different countries. These markers span a candidate region of approximately 2.1 cM. CLN3 is most prevalent in northern European populations and is especially enriched in the isolated Finnish population, with an incidence of 1:21,000. Linkage disequilibrium mapping was applied to further refine the localization of CLN3 in 27 Finnish families by using linkage disequilibrium data and information about the population history of Finland to estimate the distance of the closest markers from CLN3. CLN3 is predicted to lie 8.8 kb (range 6.3-13.8 kb) from D16S298 and 165.4 kb (132.4-218.1 kb) from D16S299. Enrichment of allele "6" at D16S298 (on 96% of Finnish and 92% of European CLN3 chromosomes) provides strong evidence that the same major mutation is responsible for Batten disease in Finland as in most other European countries and that it is therefore not a Finnish mutation. Genealogical studies show that Batten disease is widespread throughout the densely populated regions of Finland. The ancestors of two Finnish patients carrying rare alleles "3" and "5" at D16S298 in heterozygous form originate from the southwestern coast of Finland, and these probably represent other foreign mutations. Analysis of the number and distribution of CLN3 haplotypes from 12 European countries provides evidence that more than one mutation has arisen in Europe.     

Beta-lactamase genes of Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae: cloning and expression in Streptomyces lividans

Genes encoding extracellular beta-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The beta-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi beta-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The beta-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the beta-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three beta-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi beta-lactamases exhibited a 10-100-times lower activity in S. lividans, whereas the S. fradiae beta-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.     

Beneficial mutations, hitchhiking and the evolution of mutation rates in sexual populations

Natural selection acts in three ways on heritable variation for mutation rates. A modifier allele that increases the mutation rate is (i) disfavored due to association with deleterious mutations, but is also favored due to (ii) association with beneficial mutations and (iii) the reduced costs of lower fidelity replication. When a unique beneficial mutation arises and sweeps to fixation, genetic hitchhiking may cause a substantial change in the frequency of a modifier of mutation rate. In previous studies of the evolution of mutation rates in sexual populations, this effect has been underestimated. This article models the long-term effect of a series of such hitchhiking events and determines the resulting strength of indirect selection on the modifier. This is compared to the indirect selection due to deleterious mutations, when both types of mutations are randomly scattered over a given genetic map. Relative to an asexual population, increased levels of recombination reduce the effects of beneficial mutations more rapidly than those of deleterious mutations. However, the role of beneficial mutations in determining the evolutionarily stable mutation rate may still be significant if the function describing the cost of high-fidelity replication has a shallow gradient.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.     

Mapping of recognition sites for the restriction endonuclease from Escherichia coli K12 on bacteriophage PM2 DNA.

Several mutations in gene B of phage S13 appear to shorten the B protein by elimination of an N-terminal fragment, without destroying the B protein function. The shortened B protein resulting from each of these mutations can block the unique DNA-nicking properties of the S13 gene A protein. Because of the block in gene A function, normal gene B protein may have a function in phage DNA synthesis in addition to its known role in catalyzing capsid assembly.From gel electrophoresis the mutant B protein is estimated to be shorter than the normal S13 B protein by 1720 ± 70 daltons and is therefore believed to be an internal reinitiation fragment. The reinitiated fragments are functional and are made in about twice the amount of the normal B protein.The phage mutants which yield the reinitiation fragments are double mutants, each phage containing the same gene B nonsense mutation and each appearing to contain a different compensating gene B mutation. Various data support the assumption that the compensating mutations are frame-shifts, including the fact that suppression does not restore the normal-sized B protein. The reinitiation is assumed to occur at a pre-existing out-of-phase initiator codon, near the nonsense triplet; the correct reading frame would then be restored by each of the several different compensating mutations.The position of the normal S13 B protein in the gel electrophoresis pattern has been located both by elimination and shifting of the B peak, using appropriate amber mutants. The molecular weight of the S13 B protein is about 17,200, and is 2100 daltons less than the B protein of phage φX174; the S13 B protein can nevertheless substitute for the φX 174 B protein. Thus substantial portions of the B protein can be deleted without destroying its function.     

Equilibrium processes cannot explain high levels of short- and medium-range linkage disequilibrium in the domesticated grass Sorghum bicolor

Patterns of linkage disequilibrium (LD) are of interest because they provide evidence of both equilibrium (e.g., mating system or long-term population structure) and nonequilibrium (e.g., demographic or selective) processes, as well as because of their importance in strategies for identifying the genetic basis of complex phenotypes. We report patterns of short and medium range (up to 100 kb) LD in six unlinked genomic regions in the partially selfing domesticated grass, Sorghum bicolor. The extent of allelic associations in S. bicolor, as assessed by pairwise measures of LD, is higher than in maize but lower than in Arabidopsis, in qualitative agreement with expectations based on mating system. Quantitative analyses of the population recombination parameter, rho, however, based on empirical estimates of rates of recombination, mutation, and self-pollination, show that LD is more extensive than expected under a neutral equilibrium model. The disparity between rho and the population mutation parameter, , is similar to that observed in other species whose population history appears to be complex. From a practical standpoint, these results suggest that S. bicolor is well suited for association studies using reasonable numbers of markers, since LD typically extends at least several kilobases but has largely decayed by 15 kb.     

A deletion analysis of lambda hybrid phage carrying the US region of Herpes virus type 1 (Patton). II. Construction of an SmaI map

The 15.4 kb EcoRI-H fragment of Herpes simplex virus type 1 (HSV-1) strain Patton, which contains the entire short unique (US) region, has been cloned in bacteriophage lambda. The fragment contains a terminal redundancy of about 900 bp that represents the S region terminal-repeat sequences. The restriction enzyme SmaI cleaves the EcoRI-H fragment at more than 30 sites. We have constructed an SmaI map of this fragment using thirteen isolates of lambda gtWES hybrid bacteriophage that carry various deletions of the EcoRI-H fragment.     

Haplotypes and mutations in Wilson disease.

Wilson disease is a disorder of copper transport, resulting in neurological and hepatic damage due to copper toxicity. We have recently identified > 20 mutations in the copper-transporting ATPase defective in this disease. Given the difficulties of searching for mutations in a gene spanning > 80 kb of genomic DNA, haplotype data are important as a guide to mutation detection. Here we examine the haplotypes associated with specific mutations. We have extended previous studies of DNA haplotypes of dinucleotide-repeat polymorphisms (CA repeats) in the Wilson disease region to include an additional marker, in 58 families. These haplotypes, combining three markers (D13S314, D13S316, and D13S301), are usually specific for each different mutation, even though highly polymorphic CA repeat markers have been used. Haplotypes, as well as their accompanying mutations, differ between populations. In the patients whom we have studied, the haplotype data indicate that as many as 20 mutations may still be unidentified. The use of the haplotypes that we have identified provides an important guide for the identification of known mutations and can facilitate future mutation searches.     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.     

Linkage disequilibrium between mutation and RFLP haplotype at the phenylalanine hydroxylase locus in the German population

Summary Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase (PAH) locus have been determined in 60 German families with PAH deficiency. Similar to the Danish population, about 90% of the mutant alleles are confined to four distinct haplotypes. There are however, differences in the frequency distributiion of these haplotypes among the mutant alleles between the two populations. Using an oligonucleotide probe for the splicing mutation associated with mutant haplotype 3 in the Danish population, a tight association between the mutation and the RFLP haplotype has also been observed in Germany. The results provide strong evidence that the splicing mutation occurred on a haplotype 3 chromosome and that the mutant allele has spread into different populations smong Caucasians.     

Genetic profile of cosmopolitan populations: effects of hidden subdivision

Natural populations of many organisms exhibit excess of rare alleles in comparison with the predictions of the neutral mutation hypothesis. It has been shown before that either a population bottleneck or the presence of slightly deleterious mutations can explain this phenomenon. A third explanation is presented in this work, showing that hidden subdivision within a population can also lead to an excess of rare alleles in the total population when the expectations of the neutral model are based on the allele frequency profile of the entire population data. With two examples (mitochondrial DNA-morph distribution and isozyme allele frequency distributions), it is shown that most cosmopolitan human populations exhibit excess of rare as well as total allele counts, when these are compared with the expectations of the neutral mutation hypothesis. The mitochondrial data demonstrate that such excesses can be detected from genetic variation at a single locus as well, and this is not due to stochastic error of allele frequency distributions. Contrast of the present observations with the allele frequency profiles in agglomerated tribal populations from South and Central America shows that even when the neutral expectations hold for individual subpopulations, if all subpopulations are grouped into a single population, the pooled data exhibit an excess of total number of alleles that is mainly due to the excess of rare alleles. Therefore, a primary cause of the excess number of rare alleles could be the hidden subdivision, and the magnitude of the excess indicates the extent of substructuring. The two components of hidden subdivision are: 1) Number of subpopulations, and 2) the average genetic distance among them. The implications of this observation in estimating mutation rate are discussed indicating the difficulties of comparing mutation rates from different population surveys.