Biomedical subjects

R M Harding

Publications and source records attributed to R M Harding.

At least 37 records · Page 2Linked to original sources

PubMed1997

Nicking and joining activity of banana bunchy top virus replication protein in vitro.

The major open reading frame of banana bunchy top virus (BBTV) DNA-1 encodes a putative replication initiation protein (Rep). In vitro, a fusion protein of BBTV Rep linked to a maltose-binding protein exhibited both site-specific nicking and joining activities. These activities were dependent on the presence of Mg2+ or Mn2+, but did not require ATP. The fusion protein specifically cleaved ssDNA between bases +7 and +8 of a conserved nonanucleotide loop sequence which is present in the virion-strand of the stem-loop common region of each BBTV component. During this reaction, the fusion protein became covalently attached to the 5' end of the 3'cleavage product. After the nicking reactions, the fusion protein was also capable of catalysing the joining of two nicked ssDNA fragments in a site-specific manner. Based on these activities, BBTV Rep would appear to be very similar to the Rep proteins of the geminiviruses.

Cations, Divalent↗

PubMed1997

Archaic African and Asian lineages in the genetic ancestry of modern humans.

A 3-kb region encompassing the beta-globin gene has been analyzed for allelic sequence polymorphism in nine populations from Africa, Asia, and Europe. A unique gene tree was constructed from 326 sequences of 349 in the total sample. New maximum-likelihood methods for analyzing gene trees on the basis of coalescence theory have been used. The most recent common ancestor of the beta-globin gene tree is a sequence found only in Africa and estimated to have arisen approximately 800,000 years ago. There is no evidence for an exponential expansion out of a bottlenecked founding population, and an effective population size of approximately 10,000 has been maintained. Modest differences in levels of beta-globin diversity between Africa and Asia are better explained by greater African effective population size than by greater time depth. There may have been a reduction of Asian effective population size in recent evolutionary history. Characteristically Asian ancestry is estimated to be older than 200,000 years, suggesting that the ancestral hominid population at this time was widely dispersed across Africa and Asia. Patterns of beta-globin diversity suggest extensive worldwide late Pleistocene gene flow and are not easily reconciled with a unidirectional migration out of Africa 100,000 years ago and total replacement of archaic populations in Asia.

Africa↗

PubMed1996

Sequence diversity in the NIb coding region of eight sugarcane mosaic potyvirus isolates infecting sugarcane in Australia.

We have sequenced the NIb coding region of sugarcane mosaic potyvirus strain SC (SCMV-SC) and eight field isolates of SCMV from Australia. This region comprised 1563 nucleotides and encoded a putative protein of 521 amino acids containing the consensus motif GDD. The protease cleavage sites between the NIa/NIb and the NIb/coat protein were found to be Q/C and Q/A, respectively. The SCMV sequences were most similar to sorghum mosaic potyvirus with identities of 70% and 78% at the nucleotide and amino acid levels, respectively. When the sequences were compared to each other, there was a maximum of 3.3% variation between isolates at the nucleotide level and a maximum of 0.8% at the amino acid level. Phylogenetic analysis of the sequences indicated the field isolates were grouped according to their geographical location. The SCMV sequence with most homology to all other isolates has been selected to generate constructs for replicase-mediated resistance.

Amino Acid Sequence↗

PubMed1996

An ancient common origin of aboriginal Australians and New Guinea highlanders is supported by alpha-globin haplotype analysis.

The origins of aboriginal Australians and their relationship with New Guineans and neighboring Southeast Asians remains controversial. We have studied the alpha-globin haplotype composition of an aboriginal tribe from central Australia, to address some of the ambiguities of previous studies. Australians have a haplotype repertoire that is shared with New Guinea highlanders, a fact that strongly supports a common origin of these two populations. Further, Australians and New Guinea highlanders have a different set of alpha haplotypes from Southeast Asians and a lower genetic diversity. This, coupled with the presence of many locally specific central Australian haplotypes, suggests that much of the original diversity was lost in a population bottleneck prior to or during the early colonization of Sahul and that subsequent recovery of diversity has been accompanied by the generation of new haplotypes. These conclusions contrast with some previous genetic studies suggesting links between Australians, coastal New Guineans, and present-day Southeast Asians. Much of this discrepancy appears to be due to more recent Southeast Asian admixture on the north coast of Australia.

Australia↗

PubMed1995

Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

Amino Acid Sequence↗

PubMed1995

The genome organization of banana bunchy top virus: analysis of six ssDNA components.

We have cloned, sequenced and analysed an additional five circular ssDNA components of banana bunchy top virus (BBTV) which we have called components 2, 3, 4, 5 and 6. These components were present in all BBTV infections tested. Four of these components (components 3, 4, 5 and 6) had one large open reading frame (ORF) in the virion sense located 3' of a stem-loop structure. Each ORF had a potential TATA box and one or two potential polyadenylation signals associated with it and each polyadenylation signal had an associated GC-rich region containing the trinucleotide sequence TTG. A number of ORFs were identified in component 2 but none of these had appropriately located potential TATA boxes and polyadenylation signals associated with them. None of the ORF amino acid sequences nor the full DNA sequences of any of the components had significant sequence identity with any known protein or nucleic acid sequences. However, the ORF of component 4 encoded a 30 residue hydrophobic domain which may indicate that this ORF encoded a transmembrane protein. Further, the ORFs of components 3 and 5 potentially encoded proteins of about 20 kDa, the size of the BBTV coat protein. There were two regions of sequence identity between the five components described here and the previously described component 1. Each component contained a conserved stem-loop structure and a nonanucleotide potential TATA box which was 5' of the large virion-sense ORF in five of the components. The stem-loop structures were incorporated in a common region (CR-SL) of 69 nucleotides which was 62% identical between components. All six BBTV components also contained a major common region (CR-M) which was located 5' of the CR-SL in each component, in the non-coding region and was 76% identical over 92 nucleotides. Each CR-M contained a near-complete 16 nucleotide direct repeat and a GC-box which was similar to the rightward promoter element found in wheat dwarf geminivirus. From these results, BBTV appears to belong to an undescribed plant virus group which could also include subterranean clover stunt virus, coconut foliar decay virus, faba bean necrotic yellows virus and milk vetch dwarf virus.

Amino Acid Sequence↗

PubMed1995

Movement and transmission of banana bunchy top virus DNA component one in bananas.

The systemic movement and replication of banana bunchy top virus (BBTV) DNA component one were investigated. Strand-specific RNA probes and PCR were used to indicate the presence of the virus in various parts of infected banana plants during infection on the basis of dsDNA replicative intermediates of BBTV. The strand-specific probes were not only able to detect the presence of the virus but also gave an indication of where the virus replicated. The results using both the virion sense and complementary to virion sense specific probes were essentially the same indicating that BBTV initially replicated for a short period at the site of inoculation, and subsequently moved down the pseudostem to the basal meristematic region and ultimately into the roots and newly formed leaves. The virus was detected in the leaves formed prior to inoculation after 21 days using PCR but was not detected by the RNA probes. This indicated that the virus had the ability to move into these leaves but may not have replicated or accumulated to significant levels. The appearance of multimeric forms of BBTV suggested that the virus may have replicated via a rolling circle mechanism. Additionally, BBTV DNA component one did not appear to replicate in its aphid vector, Pentalonia nigronervosa.

Base Sequence↗

PubMed1995

High diversity of alpha-globin haplotypes in a Senegalese population, including many previously unreported variants.

RFLP haplotypes at the alpha-globin gene complex have been examined in 190 individuals from the Niokolo Mandenka population of Senegal: haplotypes were assigned unambiguously for 210 chromosomes. The Mandenka share with other African populations a sample size-independent haplotype diversity that is much greater than that in any non-African population: the number of haplotypes observed in the Mandenka is typically twice that seen in the non-African populations sampled to date. Of these haplotypes, 17.3% had not been observed in any previous surveys, and a further 19.1% have previously been reported only in African populations. The haplotype distribution shows clear differences between African and non-African peoples, but this is on the basis of population-specific haplotypes combined with haplotypes common to all. The relationship of the newly reported haplotypes to those previously recorded suggests that several mutation processes, particularly recombination as homologous exchange or gene conversion, have been involved in their production. A computer program based on the expectation-maximization (EM) algorithm was used to obtain maximum-likelihood estimates of haplotype frequencies for the entire data set: good concordance between the unambiguous and EM-derived sets was seen for the overall haplotype frequencies. Some of the low-frequency haplotypes reported by the estimation algorithm differ greatly, in structure, from those haplotypes known to be present in human populations, and they may not represent haplotypes actually present in the sample.

Genetic Variation↗

PubMed1994

Molecular and population genetic analysis of allelic sequence diversity at the human beta-globin locus.

Allelic sequence polymorphism at the beta-globin locus was investigated in a group of 36 Melanesians. A 3-kilobase fragment containing the gene and its flanking regions was sequenced in 60 normal (beta A) and 12 thalassemic (intron 1, position 5, G-->C) chromosomes. Haplotype relationships between linked polymorphisms were derived by allele-specific PCR amplification and sequencing. Seventeen nucleotide polymorphisms and 2 length variants were identified, and these sites segregated as 17 sequence haplotypes in the normal chromosomes. This haplotype diversity is higher than that expected on the basis of the nucleotide polymorphism observed and is probably due to recombination and gene conversion. Nucleotide diversity at synonymous sites in the sample is 0.14%, suggesting an average age of sequence divergence of approximately 450,000 years, consistent with that expected for a neutrally evolving human nuclear locus.

Alleles↗

PubMed1994

Evidence that banana bunchy top virus has a multiple component genome.

A 93 nucleotide sequence was found to be strongly conserved between two ssDNA genomic components of banana bunchy top virus (BBTV). Two outwardly extending degenerate primers were designed from this sequence and used in a polymerase chain reaction (PCR) with DNA extracted from purified BBTV virions. PCR amplified products consisting of at least seven distinct bands all approximately 1 kb and possibly representing full-length BBTV dsDNA were resolved. The PCR amplified products were cloned and the clones screened by restriction enzyme analysis. Four distinct restriction analysis groups were identified. These results confirm that the genome of BBTV contains at least five components and that it belongs to a previously undescribed group of plant viruses which may also contain subterranean clover stunt virus.

Base Sequence↗

PubMed1994

Detection and characterization of pangola stunt Fijivirus from Australia using cloned cDNA probes.

Four cDNA clones were generated from the genomic dsRNA of an Australian isolate of pangola stunt Fijivirus (PaSV). Each clone hybridized with nucleic acid extracts from PaSV infected plants but not healthy plants. Further, each clone hybridized with more than one segment of the PaSV dsRNA genome. One clone was used to demonstrate that homology existed between the Australian isolate of PaSV and a South American isolate of PaSV although the isolates differed in the sizes of the genomic dsRNAs and in the vector species. The clone also hybridized with some segments of the maize rough dwarf Fijivirus genome.

Australia↗

PubMed1994

Evidence for two groups of banana bunchy top virus isolates.

Banana bunchy top virus (BBTV) DNA component 1 from isolates from 10 different countries was cloned and sequenced and the sequences were aligned and compared. This analysis indicated two groups: the South Pacific group (isolates from Australia, Burundi, Egypt, Fiji, India, Tonga and Western Samoa) and the Asian group (isolates from the Philippines, Taiwan and Vietnam). The mean sequence difference within each group was 1.9 to 3.0% and between isolates from the two groups was approximately 10%, but some parts of the sequences differed more than others. However, the protein encoded by the major open reading frame, which is probably a replicase, differed by approximately 5%. The region from the beginning of the stem-loop sequence to the potential TATA box was identical in all isolates except for a two nucleotide change in the Western Samoan isolate and a single change in that of the NSW isolate. These results, together with other evidence, suggest that BBTV has spread to bananas after the initial movement of bananas from the Asian Pacific regions to Africa and the Americas.

Africa↗

PubMed1994

Portable peak flow meters: physical characteristics, influence of temperature, altitude, and humidity.

Little is known about the linearity of portable peak flow meters, or about physical gas factors affecting peak expiratory flow (PEF) readings. We therefore tested five portable peak flow meters of three types in an altitude chamber (sea level to 5,500 m) and in a climate chamber at sea level (7-37 degrees C) to determine the influence of the physical conditions of the gas on the reading of the meters. The nonlinear response of the variable orifice meters was confirmed and, when this was corrected for, the readings of these meters were found to be significantly reduced by higher altitude and lower temperature. The readings from a turbine type of peak flow meter were not affected by altitude but were reduced at low temperature. A mathematical model for the variable orifice meters could correct for both their nonlinear behaviour and the effect of gas density (altitude, temperature and humidity). The model showed that correction is not necessary for the differences in gas conditions between calibration and taking of measurements under normal laboratory conditions. All the meters tested had impedances higher than recommended (0.05 kPa.l-1.s) and this may influence PEF at high flows. The mean uncorrected PEF of six healthy subjects when measured with a Mini Wright peak flow meter at sea level and at 3,000 m fell by 5%, but the mean corrected PEF increased by 12%. This increase in PEF was about 60% of that predicted for fully density-dependent flow and agreed with the findings of other similar studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Demographic reductions and genetic bottlenecks in humans: minisatellite allele distributions in Oceania.

Polynesians have lower heterozygosities at minisatellite VNTR (Variable Number of Tandem Repeat) loci than have Melanesians; this has been taken as evidence of population-size bottlenecks during the colonisation of Polynesia. We have analysed the allelic distribution of several minisatellite loci in the population of Rapa, a Polynesian island that is known to have undergone a demographic reduction of approximately 95% since first contact with European explores 200 years ago, leaving a surviving population of 120. We found that the minisatellite diversity of this population does not differ significantly from that of other Polynesian populations, and appears consistent with the neutral expectation of diversity assuming the infinite alleles model. This suggests that the demographic crisis that Rapa underwent did not perturb the allele distribution to the extent that the tests used here could detect. Thus we cannot say that a demographic change of this magnitude constitutes a genetic bottleneck detectable at these loci. The reduced diversity seen in Polynesia must therefore be explained either by more severe bottlenecks as might be expected during colonisation, or else by other causes.

Alleles↗

PubMed1993

Why are some genetic diseases common? Distinguishing selection from other processes by molecular analysis of globin gene variants.

Various processes (selection, mutation, migration and genetic drift) are known to determine the frequency of genetic disease in human populations, but so far it has proved almost impossible to decide to what extent each is responsible for the presence of a particular genetic disease. The techniques of gene and haplotype analysis offer new hope in addressing this issue, and we review relevant studies of three haemoglobinopathies: sickle cell anaemia, and alpha and beta thalassaemia. We show how for each disease it is possible to recognize a pattern of regionally specific mutations, found in association with one or a few haplotypes, that is best explained as the result of selection; other patterns are due to population migration and genetic drift. However, we caution that such conclusions can be drawn in special circumstances only. In the case of the haemoglobinopathies it is possible because a selective agent (malaria) was already suspected, and the investigations could be carried out in relatively genetically homogenous populations whose migratory histories are known. Moreover, some data reviewed here suggest that gene conversion and the haplotype composition of a population may affect the frequency of a mutation, making interpretation of gene frequencies difficult on the basis of standard population genetics theory. Hence attempts to use the same approaches with other genetic diseases are likely to be frustrated by a lack of suitably untrammelled populations and by difficulties accounting for poorly understood genetic processes. We conclude that although this combination of molecular and population genetics is successful when applied to the study of haemoglobinopathies, it may not be so easy to apply it to the study of other genetic diseases.

Africa, Northern↗

PubMed1993

The population genetics of the haemoglobinopathies.

The haemoglobinopathies are the commonest single gene disorders known, and are so common in some regions of the world that the majority of the population carries at least one genetic abnormality affecting the structure or synthesis of the haemoglobin molecule. The prevalence of the common haemoglobinopathies (the alpha- and beta-thalassaemias, HbS, HbC and HbE) is almost certainly a result of the protection they provide against malaria, as the epidemiological evidence reviewed in this chapter shows. World-wide, the distributions of malaria and the common haemoglobinopathies largely overlap, and micro-epidemiological surveys have confirmed the close relationship between the disorders. However, there are complications to this picture which appear to undermine the malaria hypothesis. First, in some areas, malaria and haemoglobinopathies are not coincident. Second, the malaria hypothesis does not easily explain why no two regions of the world have the same haemoglobinopathy or combination of haemoglobinopathies. The majority of mutations have arisen only once and are regionally specific. By using molecular characterization of mutations and the analysis of haplotypes on haemoglobinopathy-bearing chromosomes it is possible to show how a combination of selection by malaria, genetic drift and population movements can explain the first complication. In order to explain the second, we have argued that malaria selection has operated relatively recently on human populations (within the last 5000 years). The present distribution is then seen as the result of selection elevating sporadic mutations in local populations. In the absence of sufficient gene flow to spread all mutations to all populations, the consequence is a patchwork distribution of haemoglobinopathies. Given time, we would expect the mutations that protect and do not compromise the health of their carriers to become widely disseminated, but it is likely that human intervention will alter this process of natural selection.

Africa↗

PubMed1993

A computer simulation study of VNTR population genetics: constrained recombination rules out the infinite alleles model.

Extensive allelic diversity in variable numbers of tandem repeats (VNTRs) has been discovered in the human genome. For population genetic studies of VNTRs, such as forensic applications, it is important to know whether a neutral mutation-drift balance of VNTR polymorphism can be represented by the infinite alleles model. The assumption of the infinite alleles model that each new mutant is unique is very likely to be violated by unequal sister chromatid exchange (USCE), the primary process believed to generate VNTR mutants. We show that increasing both mutation rates and misalignment constraint for intrachromosomal recombination in a computer simulation model reduces simulated VNTR diversity below the expectations of the infinite alleles model. Maximal constraint, represented as slippage of single repeats, reduces simulated VNTR diversity to levels expected from the stepwise mutation model. Although misalignment rule is the more important variable, mutation rate also has an effect. At moderate rates of USCE, simulated VNTR diversity fluctuates around infinite alleles expectation. However, if rates of USCE are high, as for hypervariable VNTRs, simulated VNTR diversity is consistently lower than predicted by the infinite alleles model. This has been observed for many VNTRs and accounted for by technical problems in distinguishing alleles of neighboring size classes. We use sampling theory to confirm the intrinsically poor fit to the infinite alleles model of both simulated VNTR diversity and observed VNTR polymorphisms sampled from two Papua New Guinean populations.

Alleles↗

PubMed1993

Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene.

One DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component-predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.

Amino Acid Sequence↗