Fine mapping of a gene for non-pollen type thermosensitive genic male sterility in rice (Oryza sativa L.).

The thermo-sensitive genic male sterility (TGMS) lines play a crucial role in two-line hybrid rice production. For a practical TGMS line, the stability of male sterility is one of the most important technical indicators. In this study, XianS, a spontaneous mutant with stable male sterility from an indica rice cultivar Xianhuangzhan, was classified as a non-pollen type TGMS line. The critical non-pollen sterility point temperature of XianS was determined as 27 degrees C. Genetic analysis demonstrated that the non-pollen sterility in XianS was controlled by a single recessive gene. Using SSR markers and bulked segregant analysis, the TGMS gene in XianS was fine mapped to a 183 kb interval between RMAN81 and RMX21 on chromosome 2. Two markers, 4039-1 and RMX14 completely cosegregated with this gene. Allelism test indicated that the non-pollen phenotype in seven non-pollen type TGMS lines from different sources, XianS, AnnongS-1, Q523S, Q524S, N28S, G421S, and Q527S is caused by the same TGMS gene. Although the location of TGMS gene in XianS is close to the gene OsNAC6, a previously identified candidate gene of tms5 in AnnongS-1, the sequence of OsNAC6 and its promoter region was identical in TGMS line XianS, AnnongS-1, and wild-type Xianhuangzhan. These results suggest that the non-pollen type TGMS trait probably be controlled by the same TGMS gene in different TGMS rice lines, but its real candidate gene still need to be further studied and identified.

Molecular mapping of two reverse photoperiod-sensitive genic male sterility genes (rpms1 and rpms2) in rice (Oryza sativa L.).

The reverse photoperiod-sensitive genic male sterility (PGMS) and thermo-sensitive genic male sterility (TGMS) lines have an opposite phenotype compared with normal PGMS and TGMS lines widely used by the two-line system in current hybrid rice seed production. Thus, the application of reverse PGMS and TGMS lines can compensate PGMS and TGMS lines in hybrid rice production. YiD1S is a reverse PGMS line, in which pollen fertility is mainly regulated by day-length, but also influenced by temperature. Genetic analysis indicated that male sterility of YiD1S was controlled by two recessive major genes. An F(2) population from a cross between YiD1S and 8528 was developed and used for molecular mapping of the two reverse PGMS genes which were first named rpms1 and rpms2. Both simple sequence repeat (SSR) markers and bulked segregant analysis (BSA) were used in this study. As a result, one reverse PGMS gene (rpms1) was mapped to the interval between SSR markers RM22980 (0.9 cM) and RM23017 (1.8 cM) on chromosome 8. Eight SSR markers, YDS818, RM22984, RM22986, RM22997, YDS816, RM23002, RM339 and YDS810 completely co-segregated with the rpms1 gene. Another reverse PGMS gene (rpms2) was mapped to the interval between SSR markers RM23898 (0.9 cM) and YDS926 (0.9 cM) on chromosome 9. The physical mapping information from publicly available resources shows that the rpms1 and rpms2 loci are located in a region of 998 and 68 kb, respectively. The analysis based on marker genotypes showed that the effect of rpms1 was slightly larger than that of rpms2 and that the two genes interacted in controlling male sterility.

In vitro studies on the maintenance of transcription-induced stress by histones and polyamines.

Several factors were evaluated to determine their role in facilitating the presence of transcription-induced stresses in a circular DNA. Transcription was done with T7 RNA polymerase in the presence of E. coli topoisomerase I and closed circular DNA. Positive stress was observed in hypotonic conditions or when one of the polyamines, spermidine or spermine, were present. Polycations such as polylysine, polyarginine, histone H1, histones H2A-H2B, and protamine were observed to induce minimal positive stress. It is known that polyamines influence DNA structure by causing both self-association and sequence-specific structural alterations (polyamine-induced localized bending). Experimental evidence indicates that the likely cause of the positive stress is the induced bending. In order to evaluate protein-mediated bending, transcription was done on nucleosomes. A minimum of three nucleosomes on a DNA of 6055 bp was sufficient to generate very high levels of positive stress. Histones H3-H4 in the absence of H2A-H2B were responsible for this effect. Since these histones by themselves are able to maintain negative coils on DNA, it is concluded that protein-mediated bending is yet another mechanism for placing rotational restriction on DNA. The bending of DNA by either polyamines or histones is an effective mechanism for promoting transcription-induced stresses at physiological ionic strength.

Measurement of the frequency of histone displacement during the in vitro transcription of nucleosomes: RNA is a competitor for these histones.

Transcription through tandemly arranged nucleosomes was studied to determine the frequency at which the nucleosomes would disrupt and cause displacement of the associated histones to a competitor DNA. In order to more effectively preserve topological effects, the template that was used in the in vitro transcription system was a large covalently, closed circular plasmid (8.9 kb). The plasmid contained two promoters for T7 RNA polymerase, each separated by 4.4 kb, and transcription was done in the presence of topoisomerase I at physiological ionic strength. Nucleosome disruption was observed at an approximate frequency of 1 in 4 nucleosomes such that after several rounds of transcription on the plasmid 80% of the nucleosomes were disrupted. Unexpectedly, all four histones were found associated with the RNA rather than the competitor DNA. The histones bound the competitor DNA only after removal of the RNA by RNase A treatment. By analyzing the topological state of the competitor DNA, it was observed that the majority of the histones that were displaced from the RNA were able to re-form nucleosomes. Additional experiments were done to determine the reasons for the preferential binding of histones to the newly synthesized RNA. It was found that the large molecular weight RNA binds histones with an approximate 100-fold greater affinity relative to DNA when at physiological ionic strength. Within the cell, this high-affinity binding would be expected to require cellular mechanisms to regulate the interaction of RNA with histones. The relatively high frequency of displacement of all four histones during transcription is higher than what is observed in vivo and suggests that additional factors are needed to regulate this displacement. These observations are discussed and compared with previous studies that have examined the process of transcription through nucleosomes.

Effects of spermine and its cytotoxic analogs on nucleosome formation on topologically stressed DNA in vitro.

We investigated the effects of the polyamine spermine and two of its cytotoxic analogs 1,11-bis(ethylamino)-4,8-diazaundecane (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-tirazanonadecane (BE-4-4-4-4) on the formation of nucleosomes on negatively and positively supercoiled DNA in vitro. Histones H2A, H2B, H3 and H4 were reconstituted onto DNA to form nucleosomes and the polyamines were added either before or after histone addition. The structural state of the nucleosome was monitored by analyzing the DNA topoisomers that were present after topoisomerase I treatment. Although polyamines induced DNA aggregation to various degrees. high concentrations of topoisomerase I were able to relax the aggregated DNA and the helical pitch was found to be unaltered in the aggregates. When histones were associated with negatively coiled DNA, the polyamine-induced aggregation did not alter nucleosome structure. The induced aggregate did inhibit nucleosomal transitions when examined on positively coiled DNA. BE-4-4-4-4 was most effective and BE-3-3-3 least effective. These analogs were also extremely effective in inhibiting histone deposition onto DNA. A potential mechanism for the action of these analogs is both to inhibit histone deposition during DNA replication and also disrupt nucleosomal dynamics due to aberrant chromatin condensation. These results also suggest that BE-4-4-4-4 and BE-3-3-3 may produce their cytotoxic effect through slightly different mechanisms.