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J N Timmis

Publications and source records attributed to J N Timmis.

At least 19 recordsLinked to original sources
PubMed2004

The B chromosomes in Brachycome.

This review presents a historical account of studies of B chromosomes in the genus Brachycome Cass. (synonym: Brachyscome) from the earliest cytological investigations carried out in the late 1960s though to the most recent molecular analyses. Molecular analyses provide insights into the origin and evolution of the B chromosomes (Bs) of Brachycome dichromosomatica, a species which has Bs of two different sizes. The larger Bs are somatically stable whereas the smaller, or micro, Bs are somatically unstable. Both B types contain clusters of ribosomal RNA genes that have been shown unequivocally to be inactive in the case of the larger Bs. The large Bs carry a family of tandem repeat sequences (Bd49) that are located mainly at the centromere. Multiple copies of sequences related to this repeat are present on the A chromosomes (As) of related species, whereas only a few copies exist in the A chromosomes of B. dichromosomatica. The micro Bs share DNA sequences with the As and the larger Bs, and they also have B-specific repeats (Bdm29 and Bdm54). In some cases repeat sequences on the micro Bs have been shown to occur as clusters on the A chromosomes in a proportion of individuals within a population. It is clear that none of these B types originated by simple excision of segments from the A chromosomes.

Chromosomes, Plant↗
PubMed2002

Characterisation of six alpha-expansin genes in Gossypium hirsutum (upland cotton).

A genomic library screen and PCR-based strategies were employed to isolate six genes with sequence similarity to a cotton fibre-specific mRNA encoding an alpha-expansin. alpha-Expansins are cell wall proteins that facilitate cell wall extension by disruption of non-covalent bonds between wall components. The characterisation and expression analysis of these six novel genes ( GhExp1-GhExp6) is described. Four of them ( GhExp3- GhExp6) are expressed within multiple tissues of the cotton plant and two ( GhExp1 and GhExp2) give rise to transcripts that are specific to the developing cotton fibre. GhExp1 transcripts are highly abundant in the fibre, while transcripts for GhExp2 are detected at a low level. Cotton fibres are highly elongated cells of the ovule epidermis, and we envisage that GhExp1 may play an important role in cell wall extension during development.

Amino Acid Sequence↗
PubMed2001

The genomic complexity of micro B chromosomes of Brachycome dichromosomatica.

A major sequence component of the micro B chromosome of Brachycome dichromosomatica (2 n=4) is the tandem repeat Bdm29, which was found by in situ hybridisation to be distributed along the entire length of the chromosome. A high copy number of this sequence does not occur as a regular feature of the A chromosomes in this species but it was found in infrequent individuals in two wild populations that were analysed. In these instances Bdm29 is localised within heterochromatic, polymorphic segments on the long arm of chromosome 1. The origin of the micro B chromosomes was investigated by determining whether they are related to this A chromosome polymorphism by simple excision and/or integration. Results obtained by using Bdm29, together with a newly isolated repeat sequence, Bdm54, and a number of other sequences known to occur on the micro B chromosome, as probes in in situ hybridisation and Southern analysis demonstrated that the formation of micro B chromosomes is a complex multistep process. The observation that the genomic organisation of the micro B chromosome is unlike anything found on the A chromosomes precludes their origin by simple excision and also indicates that micro Bs do not integrate directly into the A complement to form polymorphic heterochomatic segments.

Asteraceae↗
PubMed2000

Expression of a lipid transfer protein gene family during cotton fibre development.

There are six to eight lipid transfer protein-related genes in the allotetraploid Gossypium hirsutum genome, three of which, FSltp1, FSltp2 and FSltp3, were isolated in two genomic clones. All three genes potentially encode full-length lipid transfer proteins (LTPs), but sequence differences between these and the cotton fibre-specific cDNA previously reported, pFS6, suggest that none is likely to encode the fibre transcripts. Gene-specific RT-PCR experiments showed that expression patterns of the three new LTP genes are similar to each other, with transcripts present in all aerial cotton tissues tested, and only pFS6 transcripts are present in fibre cells at elevated levels. PCR analysis of cotton genomic DNA suggested that FSltp1 and FSltp3 are alloalleles, with FSltp3 being derived from the ancestral A genome, and FSltp1 from the D genome. The fibre-specific gene, yet to be isolated, and FSltp2 are also derived from the ancestral D genome of G. hirsutum.

Amino Acid Sequence↗
PubMed2000

Cloning and characterisation of polymorphic heterochromatic segments of Brachycome dichromosomatica.

After selective enrichment and differential hybridisation of Cot-1 DNA fractions of plants with and without polymorphic heterochromatic segments, a repetitive sequence (called Bds1) specific to the polymorphic chromosome segments of Brachycome dichromosomatica (Brachyscome dichromosomatica) was isolated. A single repeat unit of Bds1 is 92 bp long and is organised in tandem arrays at three different polymorphic segment sites on the chromosomes of cytodeme A2. Although all three sites showed extensive polymorphism between plants, the karyotypes of all analysed mitotic root cells were stable within a single plant. Electron microscopy revealed heavily condensed chromatin structures at the most obvious polymorphic site. The mechanisms that generate and maintain the observed chromosome structure polymorphisms are discussed.

Base Sequence↗
PubMed2000

Novel insertion sequence elements associated with genetic heterogeneity and phenotype conversion in Ralstonia solanacearum.

Three insertion sequences (IS) elements were isolated from the phytopathogen Ralstonia solanacearum. Southern hybridization using these IS elements as probes revealed hybridization profiles that varied greatly between different strains of the pathogen. During a spontaneous phenotype conversion event, the promoter of the phcA gene was interrupted by one of these IS elements.

Bacterial Proteins↗
PubMed1998

Specific expression of an expansin gene during elongation of cotton fibres.

A differential screening experiment resulted in the isolation of a full-length cDNA clone encoding an expansin from cotton fibres. Nucleotide and derived amino acid sequence data showed that pGhEX1 encodes an expansin of 258 amino acids, with an N-terminal signal peptide. Northern blot analysis showed that the corresponding transcript is abundant in cotton fibre cells but absent in all other tissues tested, and that the gene is developmentally regulated during fibre elongation.

Amino Acid Sequence↗
PubMed1998

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants.

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.

Arabidopsis↗
PubMed1997

A repetitive DNA sequence common to the different B chromosomes of the genus Brachycome.

Dot-like micro B chromosomes of Brachycome dichromosomatica were analysed for their sequence composition. Southern hybridization patterns of a total micro B probe to genomic DNA from plants with and without micro Bs demonstrated that the micro Bs shared sequences with the A chromosomes. In addition to telomere, rDNA and common A and B chromosome sequences, a new B-specific, highly methylated tandem repeat (Bdm29) was detected. After in situ hybridization with Bdm29 the entire micro B chromosome was labelled and clustering of the condensed micro Bs could be observed at interphase. A high number of Bdm29-like sequences were also found in the larger B chromosomes of B. dichromosomatica and in other Bs within the genus Brachycome.

Base Sequence↗
PubMed1997

Differences of histone H4 acetylation and replication timing between A and B chromosomes of brachycome dichromosomatica.

Differences are demonstrated between A (transcriptionally active) and B (transcriptionally inactive) chromosomes that are characterized by a different level of histone H4 acetylation and a different timing of DNA replication. These differences between the chromatin of A and B chromosomes were found after immunolabelling of chromsomes of Brachycome dichromosomatica with antibodies specific for different acetylated forms (lysine 5, 8, 12 and 16) of histone H4. In contrast to the A chromosomes, which are labelled brightly in their entirety, the transcriptionally inactive B chromosomes are faintly labelled with antibodies against H4Ac5 and H4Ac8. No such difference between the chromosomes is found after immunostaining with the other antibodies H4Ac12 and H4Ac16. Analysis of DNA replication timing in root-tip meristems suggests that B chromosomes are labelled late in S-phase compared with A chromosomes. After C-banding the B chromosome appeared to have a similar amount of heterochromatin to the A chromosomes.

Acetylation↗
PubMed1997

Ribosomal RNA genes specific to the B chromosomes in Brachycome dichromosomatica are not transcribed in leaf tissue.

Ribosomal RNA genes are present near the end of the short arm and, to a lesser extent, near the centromere of the B chromosomes of some populations of Brachycome dichromosomatica. The internal transcribed spacer (ITS2) was amplified by PCR from total leaf DNA using primers within the conserved regions encoding the 5.8S and 25S stable rRNA species. Comparison of PCR amplified ITS2 sequences from several individual plants without B chromosomes with corresponding sequences derived from microdissected B chromosomes revealed two consistent differences between the rDNA of A and B chromosomes. One of these differences produced an SfcI restriction site that was present only in the ITS2 of the B-chromosome rDNA. Amplification by PCR of ITS2 from total genomic DNA from plants with and without B chromosomes showed an additive relationship between the amount of PCR product containing the SfcI site and the number of B chromosomes present. Quantitative analysis indicated that the proportion of total nuclear rDNA present on a single B chromosome varied between 2 and 4% in different A chromosome backgrounds. Similar experiments, with appropriate positive and negative controls, using reverse transcriptase PCR of the equivalent region within the 40S precursor rRNA, suggested that the B-chromosome rDNA was not transcribed. Similarly, PCR of reverse transcribed total RNA from plants containing B chromosomes using primers specific for the B chromosome ITS2 was unable to detect a transcript from the B chromosome.

Base Sequence↗
PubMed1996

The molecular organisation of a B chromosome tandem repeat sequence from Brachycome dichromosomatica.

A high copy, tandemly repeated, sequence (Bd49) specific to the B chromosome and located near the centromere in Brachycome dichromosomatica was used to identify lambda genomic clones from DNA of a 3B plant. Only one clone of those analysed was composed entirely of a tandem array of the B-specific repeat unit. In other clones, the Bd49 repeats were linked to, or interspersed with, sequences that are repetitious and distributed elsewhere on the A and B chromosomes. One such repetitious flanking sequence has similarity to retrotransposon sequences and a second is similar to chloroplast DNA sequences. Of the four separate junctions analysed of Bd49-like sequence with flanking sequence, three were associated with the same A/T-rich region in Bd49 and the fourth was close to a 25 bp imperfect dyadic sequence. No novel B-specific sequences were detected within the genomic clones.

Base Sequence↗
PubMed1995

Organisation and origin of a B chromosome centromeric sequence from Brachycome dichromosomatica.

Brachycome dichromosomatica is an Australian native daisy that has two pairs of A chromosomes and up to three B chromosomes in some populations. A putative B-specific tandem repeat DNA sequence (Bd49) was isolated previously. Here we describe further characterisation of this sequence and investigate its possible origin. Southern analysis showed that all individual B chromosomes examined have highly methylated tandem repeats of Bd49 but differences in banding pattern for distinct B isolates suggested that the sequence is in a state of flux. Using in situ hybridisation, the sequence was shown to be located at the centromeric region of the B chromosome. Southern analysis of genomic DNA with Bd49 demonstrated that multiple copies of the sequence exist in the genomes of B. eriogona, B. ciliaris, B. segmentosa and B. multifida (none of which have B chromosomes) whereas other species tested (including 0B plants of B. dichromosomatica and 0B and +B B. curvicarpa and B. dentata) have few or no copies. Genomic clones and Bd49-like sequences derived by the polymerase chain reaction (PCR) were obtained from five species but determination of phylogenetic relationships within the genus and inference as to the possible origin of the B chromosome were problematic because of extensive intragenomic heterogeneity of the sequences.

Base Sequence↗
PubMed1995

Ribosomal RNA genes and the B chromosome of Brachycome dichromosomatica.

Fluorescence in situ hybridization (FISH) with biotinylated rDNA revealed the presence of an rRNA gene cluster on both the A and B chromosomes of Brachycome dichromosomatica, an Australian native ephemeral plant of the arid regions of south-eastern Australia. This species contains only two pairs of A chromosomes and up to three B chromosomes. The regular attachment of the B chromosome to a nucleolus suggests that these ribosomal RNA genes are transcribed. Southern hybridization of DNA from 0B and +B plants digested with a variety of restriction enzymes indicates that the rRNA genes on the A and B chromosomes are the same in sequence and methylation status.

Blotting, Southern↗
PubMed1992

Plastid DNA sequence homologies in the tobacco nuclear genome.

The tobacco (Nicotiana tabacum) nuclear genome contains long tracts of DNA (i.e. in excess of 18 kb) with high sequence homology to the tobacco plastid genome. Five lambda clones containing these nuclear DNA sequences encompass more than one-third of the tobacco plastid genome. The absolute size of these five integrants is unknown but potentially includes uninterrupted sequences that are as large as the plastid genome itself. An additional sequence was cloned consisting of both nuclear and plastid-derived DNA sequences. The nuclear component of the clone is part of a family of repeats, which are present in about 400 locations in the nuclear genome. The homologous sequences present in chromosomal DNA were very similar to those of the corresponding sequences in the plastid genome. However significant sequence divergence, including base substitutions, insertions and deletions of up to 41 bp, was observed between these nuclear sequences and the plastid genome. Associated with the larger deletions were sequence motifs suggesting that processes such as DNA replication slippage and excision of hairpin loops may have been involved in deletion formation.

Base Sequence↗
PubMed1991

A sequence specific to B chromosomes of Brachycome dichromosomatica.

Supernumerary B chromosomes represent one of many causes of numerical chromosome variation that exist in higher plants and animals. Sequences of DNA unique to B chromosomes of Brachycome dichromosomatica were enriched prior to cloning and resultant clones hybridizing only to plants containing B chromosomes were further investigated. Sequences of DNA that were characterised include members of a family of 176-bp tandem repeats that are specific to the B chromosomes of B. dichromosomatica, an annual Australian native plant species with only two pairs of A chromosomes and up to three dispensable B chromosomes. Sequence analysis of these six related clones indicated that some regions of the sequence are more highly conserved than others or, alternatively, that some adenine residues at the NdeII site are methylated. The repeat is homologous to DNA from Brachycome ciliaris var. languinosa but not to DNA from other related taxa growing in the vicinity of the B. dichromosomatica populations.

Base Sequence↗
PubMed1981

Divergence, differential methylation and interspersion of melon satellite DNA sequences.

Melon (Cucumis melo) satellite DNA consists of two components, Q and S, each with a buoyant density in CsCl of 1.707 g/ml, but differing by 9 degrees C in "melting" temperature. These physical properties appear to be in contradiction, since both depend on G + C content. In order to resolve this anomaly, base compositions were directly determined for isolated fractions. the low-"melting" component S contains 41.8% G + C, with 6% of C present as 5-methylcytosine, whereas Q DNA contains 54% G + C, with 41% of C methylated. Analyses of restriction site loss agreed well with the direct determinations of methylation and divergence, and indicated some clustering of methylated sites in Q DNA. Analysis of restricted main-band DNA by hydridization with RNA complementary to Q satellite DNA ("Southern transfer") showed satellite Q tandem arrays interspersed in DNA of main-band density. Sequence divergence and extent of methylation did not appear to depend on whether a repeat array was present as satellite or interspersed in main-band DNA. Hydridization in situ indicated considerable heterogeneity in the genomic proportion of the Q-DNA sequences in melon fruit nuclei, implying over- and under-representation consistent with extensive unequal recombination in satellite Q tandem arrays. The cucumber, Cucumis sativus, contains less than 8% as much Q-homologous DNA per genome as the melon, suggesting rapid evolutionary gain or loss of these tandem repeat sequences.

Base Composition↗