This paper describes the effects of hypomethylation with 5-azacytidine
(5azaC) and dihydroxypropyladenine
(DHPA) on protonemata of the moss Funaria hygrometrica Hedw.
Following treatment with 5azaC or DHPA,
hypomethylation of the EcoR II (CCA(T)GG) and Pst I
(CTGCAG) sites in ribosomal DNA (rDNA) was
confirmed using restriction enzyme analysis and Southern hybridization.
Hypomethylation of the genome had
profound effects on protonemal differentiation. Whilst apical cell organization
and cell dimensions and shape
remained unchanged, there was a marked retardation in both cytoplasmic
and nuclear differentiation.
Developmental abnormalities included: late and erratic side branch formation,
some loss of the distinction
between chloronema and caulonema, formation of aberrant buds, and loss
of the potential to form brood and
tmema cells after 5azaC treatment which in general had more profound
effects than DHPA. Cytologically,
caulonema cells were less highly polarized or unpolarized, and tended
to retain small round chloroplasts, whilst
nuclei in the hypomethylated protonema endoreduplicated to lower levels
and tended to remain more spherical.
Increased nucleolar volumes and loss of intranucleolar rDNA heterochromatin
following hypomethylation might
be the result of increased transcriptional activity of ribosomal RNA
(rRNA) genes and drug-induced DNA
decondensation, respectively. Growing hypomethylated protonema at
25°C induced extremely atypical cells and
development. This temperature sensitivity and aberrations in development
overall can be attributed to changes in
the normal patterns of gene expression brought about by hypomethylation
of gene promoter or regulator regions.