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Biomedical subjects

M A Matzke

Publications and source records attributed to M A Matzke.

At least 19 recordsLinked to original sources
PubMed1999

Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants.

Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of approximately 10(3). Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution.

Base Sequence↗
PubMed1999

Epigenetics: regulation through repression.

Epigenetics is the study of heritable changes in gene expression that occur without a change in DNA sequence. Epigenetic phenomena have major economic and medical relevance, and several, such as imprinting and paramutation, violate Mendelian principles. Recent discoveries link the recognition of nucleic acid sequence homology to the targeting of DNA methylation, chromosome remodeling, and RNA turnover. Although epigenetic mechanisms help to protect cells from parasitic elements, this defense can complicate the genetic manipulation of plants and animals. Essential for normal development, epigenetic controls become misdirected in cancer cells and other human disease syndromes.

Animals↗
PubMed1999

Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans.

Previous work has suggested that de novo methylation of plant nuclear genes can be triggered by an RNA-DNA interaction. To test whether transcription of a promoter would induce de novo methylation and silencing of unlinked genes driven by the same promoter, a chimeric 'gene' consisting of a nopaline synthase promoter (NOSpro) positioned downstream of the cauliflower mosaic virus 35S promoter (35Spro) and flanked at the 3' end by a NOS terminator (NOSter) was constructed and introduced into the genome of a plant that normally expresses an unmethylated NOSpro-neomycinphosphotransferase (nptII) gene. Transformants were tested for kanamycin resistance and NOSpro RNA synthesis. Most produced a full-length polyadenylated NOSpro RNA, which did not induce silencing or methylation at the NOSpro-nptII target gene. One, however, contained truncated non-polyadenylated NOSpro RNA; in this plant, the NOSpro-nptII gene became silenced and methylated in the NOSpro region. Molecular analysis of the NOSpro silencing locus revealed two incomplete copies of the 35Spro-NOSpro gene arranged as an inverted repeat with NOSpro sequences at the center. Reducing NOSpro transcription by crossing a 35Spro-silencing locus partially reactivated nptII gene expression and decreased NOSpro methylation at the target locus, thus implicating aberrant NOSpro RNA in this trans-silencing phenomenon.

Amino Acid Oxidoreductases↗
PubMed1999

Rapid structural and epigenetic changes in polyploid and aneuploid genomes.

Recent work with plants has demonstrated that genome instability can be triggered by a change in chromosome number arising from either whole genome duplications (polyploidy) or loss/gain of individual chromosomes (aneuploidy). This genome instability is manifested as rapid structural and epigenetic alterations that can occur somatically or meiotically within a few generations after heteroploid formation. The intrinsic instability of newly formed polyploid and aneuploid genomes has relevance for genome evolution and human carcinogenesis, and points toward recombinational and epigenetic mechanisms that sense and respond to chromosome numerical changes.

Aneuploidy↗
PubMed1998

Gene silencing in plants: relevance for genome evolution and the acquisition of genomic methylation patterns.

Transgenes often become silenced in plants because of repressive influences exerted by flanking plant DNA and/or because of interactions among multiple copies of closely linked transgenes. Repeated transgenes on different chromosomes can also interact in a way that leads to silencing and methylation, suggesting a previously unrecognized ability of unlinked homologous sequences to cross-talk in complex genomes. Non-Mendelian inheritance is a frequent consequence of these interactions because the silenced genes do not fully reactivate or lose methylation after segregating in progeny. Several examples of gene silencing in plants appear to reflect the action of genome defence system that methylates and inactivates foreign or invasive sequences such as transgenes and transposable elements. Because certain types of transposable elements are embedded in regulatory regions of plant genes and have become greatly amplified in plant genomes, they could contribute substantially to normal gene expression and to the generation of genomic methylation patterns. Polyploidy, which has been a major force in plant and vertebrate evolution, might encourage proliferation of transposable elements because genes in polyploids are duplicated and hence less susceptible to the consequences of insertional mutagenesis. Accordingly, the appearance of genome-wide methylation has often coincided with episodes of polyploidization.

DNA Methylation↗
PubMed1998

Epigenetic silencing of plant transgenes as a consequence of diverse cellular defence responses.

Linked and unlinked copies of transgenes and related endogenous genes in plants can be epigenetically silenced by homology-based mechanisms that operate at either the transcriptional or post-transcriptional level. Transcriptional inactivation is associated with promoter homology and meiotically heritable methylation. Post-transcriptional silencing requires homology in protein-coding regions and is fully reversed during meiosis. Recently, the notion that both of these processes reflect the action of different host defence systems has been strengthened: (i) Obvious parallels have emerged between promoter homology-dependent silencing/methylation of transgenes and paramutation of endogenous genes that contain transposable elements in their promoters: (ii) remarkable similarities have been observed between post-transcriptional silencing involving transgenes and natural forms of virus resistance in nontransgenic plants. These results and others implicate two distinct cellular defence responses in transgene silencing. One is active in the nucleus and is manifested by transgene methylation, a reaction that might have originated as a means to oppose the spread of transposable elements. A second line of defence resides in the cytoplasm and operates through enhanced RNA turnover, a process that might help plants overcome viral infection.

Cell Survival↗
PubMed1998

Position effects and epigenetic silencing of plant transgenes.

Nuclear processes that silence plant transgenes are being revealed by analyses of natural triggers of epigenetic modifications, particularly cytosine methylation, and by comparisons of the genomic environments of differentially expressed transgene loci. It is increasingly apparent that plant genomes can sense and respond to the presence of foreign DNA in certain sequence contexts and at multiple dispersed sites. Determining the basis of this sensitivity and how nuclear defense systems are activated poses major challenges for the future.

Cytosine↗
PubMed1996

Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity.

The promoter homology-dependent inactivation of a 35Spro-hygromycin phosphotransferase (hpt) gene, which is present at the H2 locus, by the multipurpose 271 silencing locus has been studied. The 271 locus can silence any gene under the control of the 35Spro as well as endogenous nitrite reductase (NiR) genes of tobacco because of the presence of a chimeric antisense gene (35Spro-RiN). All F1 progeny of a cross between homozygous H2 and 271 lines were sensitive to hygromycin and were chlorotic (a symptom of nitrogen deficiency). These phenotypes were accompanied by a reduction in the steady-state levels of Hyg and NiR transcripts. Transcriptional run-on experiments indicated, however, that while NiR silencing occurred post-transcriptionally, the hpt gene was inactivated at the transcriptional level; this was associated with increased methylation of the 35Spro of the hpt gene. NiR gene expression recovered uniformly to wild-type levels in first generation backcross (BC1) progeny that did not inherit the 271 locus. In contrast, hygromycin resistance was only partially and non-uniformly regained among adult BC1 plants. Moreover, substantial silencing of the hpt gene could persist into the BC2 generation. Genomic sequencing demonstrated that the meiotic heritability of hpt silencing in the absence of the 271 locus was correlated with cytosine methylation primarily at CpG and CpNpG residues. Despite this residual methylation, H2 loci weakened by an association with 271 did not acquire the ability to silence a 'naive' H2 locus. Fluorescence in situ hybridization revealed that the 271 locus was located at a telomere. The results strengthen the distinction between silencing effects involving homology restricted to coding or promoter regions, respectively. The former is a post-transcriptional process that is meiotically reversible; the latter is due to transcriptional inactivation and is associated with increased promoter methylation, which can lead to meiotically heritable reductions in target gene activity. The relevance of these data for the meiotic heritability of silencing, the non-transferability of silencing activity, and the basis of 271 silencing effects is discussed.

Antisense Elements (Genetics)↗
PubMed1996

Structural instability of a transgene locus in tobacco is associated with aneuploidy.

This paper describes molecular and cytogenetic evidence for the stability of a transgene locus that is present on the triplicated chromosome in an aneuploid tobacco line. This instability was manifested in several ways in trisomics including a major chromosome rearrangement that was detectable cytogenetically, smaller scale DNA rearrangements that occurred both germinally and somatically, and methylation/epigenetic silencing. In a deletion derivative of the locus, DNA breakpoints were found in AT-rich regions. One of these regions binds to nuclear scaffolds in vitro, suggesting a possible role for aberrant topoisomerase II cleavage in destabilization of the locus. The implications of increased chromosome instability in aneuploids for plant karyotype evolution and human carcinogenesis are discussed.

Aneuploidy↗
PubMed1996

The use of combined FISH/GISH in conjunction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco.

We have used combined fluorescent and genomic in situ hybridization (FISH/GISH) together with 4',6-diamidino-2-phenylindole (DAPI) counterstaining to determine simultaneously the chromosome integration site and subgenomic allocation of a transgene in-sert in amphidiploid tobacco (Nicotiana tabacum, 2n = 4x = 48). The procedure provides sufficient information on physical markers to identify at least 20 out of 24 chromosome pairs of two tobacco cultivars commonly used in studies on transgene expression and silencing (cv. Petit Havana SR1 and cv. Gatersleben). The chromosomes can be distinguished on the basis of diploid parental ancestry, size, morphology, the presence of rDNA loci and/or intergenomic exchanges, and the DAPI banding pattern, which is shown here for the first time for N. tabacum. From a single ISH experiment, it should now be possible in most cases to identify a tobacco chromosome carrying a transgene insert, thus permitting systematic studies of how the chromosome location of transgenes influences expression levels.

Chromosomes↗
PubMed1996

The use of combined FISH/GISH in conjunction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco.

We have used combined fluorescent and genomic in situ hybridization (FISH/GISH) together with 4',6-diamidino-2-phenylindole (DAPI) counterstaining to determine simultaneously the chromosomal integration site and subgenomic allocation of a transgene insert in amphidiploid tobacco (Nicotiana tabacum, 2n = 4 chi = 48). The procedure provides sufficient information on physical markers to identify at least 20 out of 24 chromosome pairs of two tobacco cultivars commonly used in studies on transgene expression and silencing (cv. Petit Havana SR1 and cv. Gatersleben). The chromosomes can be distinguished on the basis of diploid parental ancestry, size, morphology, the presence of rDNA loci and/or intergenomic exchanges, and the DAPI banding pattern, which is shown here for the first time for N. tabacum. From a single ISH experiment, it should now be possible in most cases to identify a tobacco chromosome carrying a transgene insert, thus permitting systematic studies of how the chromosomal location of transgenes influences expression levels.

Chromosomes↗