PubMed Health⌕ Search

Biomedical subjects

P R England

Publications and source records attributed to P R England.

12 recordsLinked to original sources
PubMed2006

Assessment of population structure by single nucleotide polymorphisms (SNPs) in goat breeds.

Single nucleotide polymorphisms (SNPs) may be used in biodiversity studies and commercial tasks like traceability, paternity testing and selection for suitable genotypes. Twenty-seven SNPs were characterized and genotyped on 250 individuals belonging to eight Italian goat breeds. Multilocus genotype data were used to infer population structure and assign individuals to populations. To estimate the number of groups (K) to test in population structure analysis we used likelihood values and variance of the bootstrap samples, deriving optimal K from a drop in the likelihood and a rise in the variance plots against K.

Animals↗
PubMed2003

Effects of seed bank disturbance on the fine-scale genetic structure of populations of the rare shrub Grevillea macleayana.

Dispersal in most plants is mediated by the movement of seeds and pollen, which move genes across the landscape differently. Grevillea macleayana is a rare, fire-dependent Australian shrub with large seeds lacking adaptations for dispersal; yet it produces inflorescences adapted to pollination by highly mobile vertebrates (eg birds). Interpreting fine-scale genetic structure in the light of these two processes is confounded by the recent imposition of anthropogenic disturbances with potentially contrasting genetic consequences: (1) the unusual foraging behaviour of exotic honeybees and 2. widespread disturbance of the soil-stored seedbank by road building and quarrying. To test for evidence of fine-scale genetic structure within G. macleayana populations and to test the prediction that such structure might be masked by disturbance of the seed bank, we sampled two sites in undisturbed habitat and compared their genetic structure with two sites that had been strongly affected by road building using a test for spatial autocorrelation of genotypes. High selfing levels inferred from genotypes at all four sites implies that pollen dispersal is limited. Consistent with this, we observed substantial spatial clustering of genes at 10 m or less in the two undisturbed populations and argue that this reflects the predicted effects of both high selfing levels and limited seed dispersal. In contrast, at the two sites disturbed by road building, spatial autocorrelation was weak. This suggests there has been mixing of the seed bank, counteracting the naturally low dispersal and elevated selfing due to honeybees. Pollination between near neighbours with reduced relatedness potentially has fitness consequences for G. macleayana in disturbed sites.

Australia↗
PubMed1996

Microsatellite polymorphisms in a wild population of Drosophila melanogaster.

Highly variable DNA polymorphisms called microsatellites are rapidly becoming the marker of choice in population genetic studies. Until now, microsatellites have not been utilized for Drosophila studies. We have identified eight polymorphic microsatellite loci in Drosophila melanogaster and used them to characterize the genetic variation in a wild population from the Tyrrell's winery in Australia. Microsatellites were isolated from a partial genomic DNA library. All microsatellites consist of (AC)n repeats ranging from n = 2 to n = 24. Six loci were assigned to chromosomal location by genetic mapping, with three loci on chromosome II, one locus on chromosome III and two loci on the X chromosome. Up to four microsatellite loci were multiplexed in the same reaction. Microsatellite variation is substantially greater than allozyme variation in the Tyrrell's Drosophila population. 80% of the microsatellite loci examined are polymorphic, compared with 28% of allozymes. The mean number of alleles per polymorphic locus is 5.2 in microsatellites compared with 3.0 in allozymes. The average observed heterozygosity of polymorphic microsatellites is 47% compared with 26% for allozymes. Microsatellite variation in Drosophila melanogaster is similar to that reported for other insects. Higher variability commends microsatellites over allozymes for genetic studies in Drosophila melanogaster.

Animals↗
PubMed1996

Microsatellite and chromosome evolution of parthenogenetic sitobion aphids in Australia.

Single-locus microsatellite variation correlated perfectly with chromosome number in Sitobion miscanthi aphids. The microsatellites were highly heterozygous, with up to 10 alleles per locus in this species. Despite this considerable allelic variation, only seven different S. miscanthi genotypes were discovered in 555 individuals collected from a wide range of locations, hosts and sampling periods. Relatedness between genotypes suggests only two successful colonizations of Australia. There was no evidence for genetic recombination in 555 S. miscanthi so the occurrence of recent sexual reproduction must be near zero. Thus diversification is by mutation and chromosomal rearrangement alone. Since the aphids showed no sexual recombination, microsatellites can mutate without meiosis. Five of seven microsatellite differences were a single repeat unit, and one larger jump is likely. The minimum numbers of changes between karyotypes corresponded roughly one-to-one with microsatellite allele changes, which suggests very rapid chromosomal evolution. A chromosomal fission occurred in a cultured line, and a previously unknown chromosomal race was detected. All 121 diverse S. near fragariae were heterozygous but revealed only one genotype. This species too must have a low rate of sexual reproduction and few colonizations of Australia.

Animals↗
PubMed1996

Low genetic variability of the koala Phascolarctos cinereus in south-eastern Australia following a severe population bottleneck.

Genotyping of koalas at CA-repeat microsatellite loci has revealed significant differences in the levels of allelic diversity (A) and expected heterozygosity (H(E)) between populations from north-eastern and south-eastern Australia. In the 10 populations studied, allelic diversity ranged from 8.0 in the Nowendoc population to 1.7 in the Kangaroo Is. population, and values of H(E) ranged from 0.831 in the Nowendoc population to 0.331 in the Kangaroo Is. population. Data from pooled populations revealed koalas from the north-eastern region had significantly higher levels of allelic diversity (A = 11.5 +/- 1.4) than those from south-eastern Australia (A = 5.3 +/- 1.0). Furthermore significantly higher heterozygosity levels were found in the north-eastern (H(E) = 0.851) vs. the south-eastern (H(E) = 0.436) regions of Australia. Following a near-extinction bottleneck in the 1920s, mainland Victorian and Kangaroo Is. koalas have been involved in an extensive program of relocations. The source populations of the relocated animals were islands in Westernport Bay, which were founded by very few individuals in the late 1800s and early 1900s. The significantly lower levels of variation between south-eastern Australian populations suggests that human intervention has had a severe effect on levels of genetic diversity in this region, and this may have long-term genetic consequences.

Alleles↗
PubMed1993

The determination of the sequences present in the shadow bands of a dinucleotide repeat PCR.

A Polymerase Chain Reaction (PCR) of a DNA sequence containing a CA repeat produces a main band but also several shadow bands that differ by 2 base pairs below the main band. In the experiments described in this paper, these shadow bands were excised from a DNA sequencing gel and directly sequenced. It was found that the sequence in the CA repeat was ambiguous. However, the sequence 5' and 3' to the CA repeat was clear and unambiguous. It is proposed that the shadow bands are generated by 2 base pair random deletions in the CA repeat region. During this process the sequence becomes 'scrambled' only in the CA repeat region. The shadow bands were shown to occur during the PCR since the genomic DNA template did not contain the shadow bands. It is probable that the shadow bands arise by slippage during the PCR. It is predicted that a thermostable DNA polymerase with a high processivity would greatly reduce the occurrence of shadow bands.

Base Sequence↗
PubMed1993

Detection of polymorphisms using thermal cycling with a single oligonucleotide on a DNA sequencing gel.

A method is described for the detection of restriction fragment length polymorphisms (RFLPs) in single copy genes in mammalian cells using one 5'-labelled oligonucleotide. This linear amplification (LA) method employs a single oligonucleotide as primer, which is extended by Taq DNA polymerase up to a restriction enzyme cleavage site. The products are arithmetically amplified by thermal cycling. The size of the products are determined by the sequence of the oligonucleotide and the position of the restriction enzyme cleavage site. Hence, an RFLP can be observed by measuring the size of the products. Polymorphisms which differ in size by a small number of base pairs, as are found in (CA)n repeats, are especially suitable for analysis by the LA procedure since the products are run on DNA sequencing gels. A number of genes were examined by the procedure and all produced a satisfactory signal including GC-rich template. It is proposed that the LA method would be suitable for large-scale genetic linkage analysis. The LA procedure has many advantages including the ability to multiplex signals under the same conditions, and lower cost since only one primer is needed.

Evaluation Studies as Topic↗
PubMed1993

Sequence-specific DNA damage using iodine-125-labeled antisense oligonucleotides.

A procedure is described that cleaves single-stranded DNA with sequence specificity. This process involves attaching a DNA damaging agent to an oligonucleotide. This oligonucleotide delivers the DNA damaging agent, iodine-125, to a specific DNA sequence by complementary hybridization. 5-[125I]Iodo-2-deoxycytidine 5'-triphosphate was enzymatically incorporated into an oligonucleotide that was designed to hybridize to a single-stranded DNA target. 125I decays by electron capture and causes breaks in the target DNA. These breaks were observed on a DNA sequencing gel. After 22 days of exposure to the 125I-labeled oligonucleotide, significant damage was observed within 1 to 2 bases of the expected site of hybridization. Densitometry showed that after 48 days the amount of damage had approximately doubled. This method facilitates easy design and testing of oligonucleotides that could potentially be used to inactivate gene expression in a wide variety of organisms.

Autoradiography↗
PubMed1992

An investigation of the sequence-specific interaction of cis-diamminedichloroplatinum(II) and four analogues, including two acridine-tethered complexes, with DNA inside human cells.

The sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II) (cisplatin) and four analogues in human (HeLa) cells was studied using Taq DNA polymerase and a linear amplification system. The primer extension is inhibited by the drug-DNA adducts, and hence the sites of these lesions can be analyzed on DNA sequencing gels. The repetitive alphoid DNA was used as the target DNA in human cells. A comparison was made between adduct formation in human cells and in purified DNA. The sequence-specific position and relative intensity of damage was similar in both systems for cisplatin, dichloro(ethylenediammine)platinum(II) (PtenCl2), and N-[3-N-(ethylenediamino)propyl]acridine-4-carboxamidedichloropl atinum(II) (4AcC3PtenCl2). However, no DNA damage could be detected in cells for trans-diamminedichloroplatinum(II) (transPt) or N-[3-N-(ethylenediamino)propyl]acridine-2-carboxamide-dichloroplat inum(II) (2AcC3PtenCl2) despite the ability of these latter analogues to damage purified DNA. Cisplatin, PtenCl2, and 4AcC3PtenCl2, which significantly damaged DNA inside cells, also show antitumor activity in mouse models. However, transPt and 2AcC3PtenCl2, which did not detectably damage DNA inside cells, did not show such antitumor activity. This correlation between intracellular DNA damaging ability and in vivo antitumor activity indicates the potential use of the human cells/Taq DNA polymerase/linear amplification technique as a convenient method for screening new cisplatin analogues for useful chemotherapeutic activity.

Acridines↗
PubMed1992

The use of Taq DNA polymerase to determine the sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II), acridine-tethered platinum(II) diammine complexes or two analogues.

cis-Diamminedichloroplatinum(II) (cisplatin) forms adducts with DNA. The sequence specificity of formation of cisplatin adducts with plasmid DNA was investigated using Taq DNA polymerase. This procedure involved the extension of an oligonucleotide primer by Taq DNA polymerase up to the cisplatin adduct. Using thermal cycling, this process is repeated many times in order to amplify the signal. The products of this linear amplification can then be examined on DNA sequencing gels, and the sequence specificity of cisplatin adduct formation can be determined to the exact base pair. In the pUC8 plasmid, the sequences that produced the most intense damage sites (as determined by densitometry) were runs of two or more Gs. Adducts could also be detected at GA, AG, and GC dinucleotides. Four other cisplatin analogues were also tested in the system. Two of these analogues contained an attached intercalating chromophore, and the strong damage with these compounds was similar to that found for cisplatin, but the medium and weak damage tended to be different. Weak damage was also detected with trans-diamminedichloroplatinum(II). With this compound, a large number of the damage sites were at the CG dinucleotide. This technique represents a simple, accurate, and quick method for determining the sequence specificity of damage for a cisplatin analogue in any DNA sequence.

Autoradiography↗
PubMed1992

Direct PCR sequencing of dystrophin polymorphic CACA alleles after purification to remove shadow bands.

A method is described that allows the sequencing of polymerase chain reaction (PCR) products containing CACA repeats. The method was tested using a DNA polymorphism that exists at the 3' end of the dystrophin gene. This polymorphism consists of a variation in the length of a CACA dinucleotide repeat. Four alleles from a total of 16 individuals were sequenced at this locus after the DNA sequence had been amplified by the PCR. Five examples of each of the common alleles were sequenced. For each allele all five sequences were the same. The only example of a rare allele was also sequenced. The PCR products of DNA sequences containing dinucleotide repeats consist of a number of bands differing by 2 bp below the most intense main band. Previously, direct sequencing of the PCR products lead to ambiguities and smearing at and above the CACA repeat. In this paper, the main PCR band was cut out of a sequencing gel and directly sequenced to give a clear DNA sequence. Our results indicate that for a particular allele, all individuals had exactly the same DNA sequence. This implies that with the appropriate choice of oligonucleotide primers, polymorphisms could be detected without electrophoresis.

Alleles↗