Mazhar Ali Sheikh - Academia.edu (original) (raw)

Papers by Mazhar Ali Sheikh

Research paper thumbnail of Gene Silencing and Homology-Dependent Gene Silencing in Arabidopsis: Genetic Modifiers and DNA Methylation

Transgenes inserted into the plant genome can become inactive (gene silencing) or result in silen... more Transgenes inserted into the plant genome can become inactive (gene silencing) or result in silencing of homologous cellular genes (homology-dependent gene silencing (HDG silencing)). In an earlier study we reported HDG silencing of chalcone synthase (CHS) in Arabidopsis. This study concerns genetic revertants of one of the CHS HDG -silencing transgenic homozygotes. Two monogenic recessive trans- acting mutations (hog1 and

Research paper thumbnail of Genetics of homology‐dependent gene silencing in Arabidopsis; a role for methylation

The Plant …, 1997

Ninety-eight independent transformed (T1) Arabidopsis plants were generated, containing additiona... more Ninety-eight independent transformed (T1) Arabidopsis plants were generated, containing additional copies of the chalcone synthase (CHS) gene. Three T2 generation families (A, B and C) were found that showed reduced anthocyanin biosynthesis, consistent with homology-dependent gene silencing of CHS. Clonal sectors of tissue showing CHS silencing were seen in the early generations. Affected individuals in family A showed only slight silencing, in family C such plants were almost completely silenced, and in family B affected individuals were intermediate. Plants homozygous for a single silencing insert were isolated from each family. Plants homozygous or hemizygous for insert A showed variable penetrance and expressivity of silencing. Self-fertilization of plants hemizygous for the B and C-inserts suggested that these CHS-silencing inserts each behave as single Mendelian dominant traits. The CHS mRNA of the C-insert homozygotes was reduced to undetectable levels. Outcrosses of B- and C-insert homozygotes to wild-type plants resulted in F1 plants that were variegated. This variegation appears to be due to expression of the CHS allele from the wild-type parent, since use of a CHS mutant, tt4, as untransformed parent resulted in uniform green F1 plants. Southern blots revealed a correlation between DNA methylation and CHS silencing. In addition, derivative plants were generated from C-insert homozygotes that had lost the silencing inserts, and these showed a partial reversion towards wild-type phenotype and methylation of the cellular CHS gene at the TT4 locus. This result suggests that the TT4 copy of CHS became methylated during the C-insert-induced silencing and retained methylation and partial silencing after the silencing T-DNA was lost.

Research paper thumbnail of Unexpected silencing effects from T-DNA tags in Arabidopsis

Research paper thumbnail of The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 Gene Codes for An S-Adenosyl-L-Homocysteine Hydrolase Required for DNA Methylation-Dependent …

The Plant Cell …, 2005

Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silenc... more Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silencing of homologous genes at unlinked sites (homology-dependent gene silencing or HDG silencing). Mutations of the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) locus relieve transcriptional gene silencing and methylation-dependent HDG silencing and result in genome-wide demethylation. The hog1 mutant plants also grow slowly and have low fertility and reduced seed germination. Three independent mutants of HOG1 were each found to have point mutations at the 39 end of a gene coding for S-adenosyl-L-homocysteine (SAH) hydrolase, and hog1-1 plants show reduced SAH hydrolase activity. A transposon (hog1-4) and a T-DNA tag (hog1-5) in the HOG1 gene each behaved as zygotic embryo lethal mutants and could not be made homozygous. The results suggest that the homozygous hog1 point mutants are leaky and result in genome demethylation and poor growth and that homozygous insertion mutations result in zygotic lethality. Complementation of the hog1-1 point mutation with a T-DNA containing the gene coding for SAH hydrolase restored gene silencing, HDG silencing, DNA methylation, fast growth, and normal seed viability. The same T-DNA also complemented the zygotic embryo lethal phenotype of the hog1-4 tagged mutant. A model relating the HOG1 gene, DNA methylation, and methylation-dependent HDG silencing is presented.

Research paper thumbnail of Transcript profiling of the hypomethylated hog1 mutant of Arabidopsis

Transcript profiling was used to look for genes that differ in expression between the SAH hydrola... more Transcript profiling was used to look for genes that differ in expression between the SAH hydrolase deficient and hypomethylated hog1-1 mutant and the parental (HOG1) line. This analysis identified a subset of gene transcripts that were up-regulated in hog1-1 plants. The majority of these transcripts were from genes located in the pericentromeric heterochromatin. About a third of the genes are annotated as transposons or having transposon homology. Subsequent experiments using Northern blots, RT-PCR and real-time RT-PCR confirmed the up-regulation of 19 of the genes and identified a set of molecular probes for genes that are up-regulated in the hog1-1 background. Six (of six genes tested) of the hog1-1 up-regulated genes are also up-regulated in the hypomethylated ddm1 mutant, three in the hypomethylated met1 mutant and three in the dcl3 mutant. The results suggest that the hypomethylation in the mutant lines may have a causal role in the up-regulation of these transcripts.

Research paper thumbnail of Gene Silencing and Homology-Dependent Gene Silencing in Arabidopsis: Genetic Modifiers and DNA Methylation

Transgenes inserted into the plant genome can become inactive (gene silencing) or result in silen... more Transgenes inserted into the plant genome can become inactive (gene silencing) or result in silencing of homologous cellular genes (homology-dependent gene silencing (HDG silencing)). In an earlier study we reported HDG silencing of chalcone synthase (CHS) in Arabidopsis. This study concerns genetic revertants of one of the CHS HDG -silencing transgenic homozygotes. Two monogenic recessive trans- acting mutations (hog1 and

Research paper thumbnail of Genetics of homology‐dependent gene silencing in Arabidopsis; a role for methylation

The Plant …, 1997

Ninety-eight independent transformed (T1) Arabidopsis plants were generated, containing additiona... more Ninety-eight independent transformed (T1) Arabidopsis plants were generated, containing additional copies of the chalcone synthase (CHS) gene. Three T2 generation families (A, B and C) were found that showed reduced anthocyanin biosynthesis, consistent with homology-dependent gene silencing of CHS. Clonal sectors of tissue showing CHS silencing were seen in the early generations. Affected individuals in family A showed only slight silencing, in family C such plants were almost completely silenced, and in family B affected individuals were intermediate. Plants homozygous for a single silencing insert were isolated from each family. Plants homozygous or hemizygous for insert A showed variable penetrance and expressivity of silencing. Self-fertilization of plants hemizygous for the B and C-inserts suggested that these CHS-silencing inserts each behave as single Mendelian dominant traits. The CHS mRNA of the C-insert homozygotes was reduced to undetectable levels. Outcrosses of B- and C-insert homozygotes to wild-type plants resulted in F1 plants that were variegated. This variegation appears to be due to expression of the CHS allele from the wild-type parent, since use of a CHS mutant, tt4, as untransformed parent resulted in uniform green F1 plants. Southern blots revealed a correlation between DNA methylation and CHS silencing. In addition, derivative plants were generated from C-insert homozygotes that had lost the silencing inserts, and these showed a partial reversion towards wild-type phenotype and methylation of the cellular CHS gene at the TT4 locus. This result suggests that the TT4 copy of CHS became methylated during the C-insert-induced silencing and retained methylation and partial silencing after the silencing T-DNA was lost.

Research paper thumbnail of Unexpected silencing effects from T-DNA tags in Arabidopsis

Research paper thumbnail of The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 Gene Codes for An S-Adenosyl-L-Homocysteine Hydrolase Required for DNA Methylation-Dependent …

The Plant Cell …, 2005

Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silenc... more Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silencing of homologous genes at unlinked sites (homology-dependent gene silencing or HDG silencing). Mutations of the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) locus relieve transcriptional gene silencing and methylation-dependent HDG silencing and result in genome-wide demethylation. The hog1 mutant plants also grow slowly and have low fertility and reduced seed germination. Three independent mutants of HOG1 were each found to have point mutations at the 39 end of a gene coding for S-adenosyl-L-homocysteine (SAH) hydrolase, and hog1-1 plants show reduced SAH hydrolase activity. A transposon (hog1-4) and a T-DNA tag (hog1-5) in the HOG1 gene each behaved as zygotic embryo lethal mutants and could not be made homozygous. The results suggest that the homozygous hog1 point mutants are leaky and result in genome demethylation and poor growth and that homozygous insertion mutations result in zygotic lethality. Complementation of the hog1-1 point mutation with a T-DNA containing the gene coding for SAH hydrolase restored gene silencing, HDG silencing, DNA methylation, fast growth, and normal seed viability. The same T-DNA also complemented the zygotic embryo lethal phenotype of the hog1-4 tagged mutant. A model relating the HOG1 gene, DNA methylation, and methylation-dependent HDG silencing is presented.

Research paper thumbnail of Transcript profiling of the hypomethylated hog1 mutant of Arabidopsis

Transcript profiling was used to look for genes that differ in expression between the SAH hydrola... more Transcript profiling was used to look for genes that differ in expression between the SAH hydrolase deficient and hypomethylated hog1-1 mutant and the parental (HOG1) line. This analysis identified a subset of gene transcripts that were up-regulated in hog1-1 plants. The majority of these transcripts were from genes located in the pericentromeric heterochromatin. About a third of the genes are annotated as transposons or having transposon homology. Subsequent experiments using Northern blots, RT-PCR and real-time RT-PCR confirmed the up-regulation of 19 of the genes and identified a set of molecular probes for genes that are up-regulated in the hog1-1 background. Six (of six genes tested) of the hog1-1 up-regulated genes are also up-regulated in the hypomethylated ddm1 mutant, three in the hypomethylated met1 mutant and three in the dcl3 mutant. The results suggest that the hypomethylation in the mutant lines may have a causal role in the up-regulation of these transcripts.