Hothead ( hth/hth ) mutants in Arabidopsis All Hothead mutations are recessive – need both mutant alleles to produce phenotype All mutant alleles contain single point mutations which lead to different restriction site digestion patterns Hothead mutant phenotypes characterized thus far:
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Hothead(hth/hth) mutants in Arabidopsis
Self-fertilized homozygous hothead mutants (hth/hth) revert
to heterozygous condition (hth/HTH) at an unusually high rate
Molecular genetic analysis of HTH
PCR-based assay demonstrating genetic reversion of hth-8. DNA was extracted from mutant and revertant progeny that were derived from the self-fertilization of a homozygous hth-8 parent, amplified with gene specific primers and digested to reveal the presence or absence of a polymorphic restriction site. Controls in the left three lanes show digest patterns derived from wild-type (HTH/HTH), heterozygous (HTH/hth) and homozygous (hth/hth) mutant plants. Photos above mutant and revertant lanes show the hth/hth mutant phenotype and the revertant phenotype.
Possible alternative explanations for results
Experiment to test seed contamination hypothesis
Can pollen derived from hth/hth mutant plants
transmit an HTH wild-type allele to progeny?
Testing for a high rate of random mutations at the HTH locus
Testing gene conversion hypothesis – Genome blot indicates HTH is a single copy gene in Arabidopsis
Genome blot showing that HTH is present in a single copy in Col (C), Ler (L) and Ws (W). Genomic DNA was digested as indicated and probed with a labelled fragment of Col DNA corresponding to the HTH coding sequence.
Examining the family of
Hothead-like genes as a
source for gene conversion
Figure 2 DNA sequences of HTH and HTL genes around the sites of mutation in hth-4,hth-8 and hth-10. The location of the mutant nucleotide is boxed, and the wild-type, nucleotide at this position is shown in blue; differences from the HTH sequence are shown in red lower-case letters. For hth-4 none of the related sequences contain the wild-type nucleotide at the site of the mutation, for hth-8 all of the other sequences contain the appropriate wild-type nucleotide, and for hth-10 some of the other sequences contain the relevant wild-type nucleotide. In all cases there are sequence differences between the HTL sequences and HTH within a few nucleotides of the site of the mutation.
Do DNA sequences changes occur in other places in the genome?
Template driven mechanism – RNA cache
hypothesis vs. reverting sequence hypothesis
Figure 1 | DNA nucleotide sequences of the hth-4, hth-8, hth-10 and er mutants in the region of the mutation, compared with wild type. Sequences of the mutants are shown in blue, with the mutated nucleotide in lower-case; the corresponding wildtype sequences are in black and the nucleotide at the site of mutation is highlighted in red. Homologous sequences that might cause the mutations to revert (RS sequences), obtained by BLAST-searching the Landsberg erecta database from www.arabidopsis.org, are shown in green, with the wild-type nucleotide in red. Reversion frequency is lower for the hth-4 allele (with 6 RS sequences of 13–15 nucleotides), than for hth-8 (with 20 RS of 13–15 nucleotides) and hth-10 (with 24 RS and 13–18 nucleotides).