Earlier onset of DNA fragmentation in leaves of wheat compared to barley and rye.

We have found extensive nucleosomal fragmentation of native DNA extracted from leaves of healthy cereal plants, as indicated by ladder patterns on agarose gels and TUNEL staining. The time of first appearance of fragmentation differed among cereals. Native DNA from the first leaf of 10-day-old plants formed a clear ladder pattern of multiples of 180 bp fragments in wheat and triticale but not in barley and oats. In one cultivar of rye a weak ladder pattern occurred but not in another. Freezing and thawing of samples before DNA extraction resulted in much more extensive DNA fragmentation in wheat but not in rye and barley, indicating that DNA-degrading enzymes are present in the cytoplasm of wheat, but not in barley and rye, at this stage. In barley, nucleosomal fragmentation was first detected in 25-day-old plants. These results indicate that programmed cell death takes place in developing leaves of young cereal plants, but that the time of onset differs among cereal species.

Geographic and altitudinal allozyme variation in sorghum (Sorghum bicolor (L.) Moench) landraces from Ethiopia and Eritrea.

The amount and distribution of genetic variation was investigated in 48 sorghum landrace accessions, representing 13 regions of origin and three adaptation zones (lowland, intermediate and highland elevation) in Ethiopia and Eritrea. Assaying 11 enzymes systems, 23 putative loci were scored for a total of 27 alleles. Nineteen loci were monomorphic and fixed for the same allele, while the remaining 4 loci, each with 2 alleles, were polymorphic across the 48 accessions. The results show significant differences in allele frequencies among the accessions, regions of origin and the adaptation zones. However, all measures of genetic variation used show that the accessions maintained much lower levels of variation than the corresponding mean values for self-pollinating crop plants, confirming previous conclusions that sorghum is depauperated in allozymic variation. The total gene diversity was 0.25, which partitioned 59% within and 41% among accessions. The latter was largely due to variation among accessions within the adaptation zones (38%), while only 3% was due to variation among accessions between the adaptation zones. Similarly, most of the total gene diversity was found within the regions of origin (80%) and within the adaptation zones (97%). Both the dendrogram constructed from NEI's unbiased genetic distance and the plot of the first two principal components distinguished three groups of regions. The level of gene flow was low among accessions, regions of origin and among accessions within adaptation zones, but high among adaptation zones. The results are discussed with emphasis on genetic resources conservation and utilization.

Genetic variation in wild sorghum (Sorghum bicolor ssp. verticilliflorum (L.) Moench) germplasm from Ethiopia assessed by random amplified polymorphic DNA (RAPD).

The extent and distribution of genetic variation in wild sorghum (Sorghum bicolor ssp. verticilliflorum (L.) Moench) collected from five different geographical regions in Ethiopia were analyzed using random amplified polymorphic DNA (RAPD) markers for 93 individuals representing 11 populations. Nine decamer primers generated a total of 83 polymorphic bands with 8-12 bands per primer and a mean of 9 bands across the 93 individuals. The amount of genetic variation among the populations (H = 0.37) and among the geographical region (H = 0.44) was low to moderate, despite the high degree of polymorphic bands per primer. Similarly, the mean genetic distance (0.08) among populations as well as among regions of origin (0.04) of the population was found to be low. The low genetic variation may be due to the reduced population size of the wild sorghum in Ethiopia because of habitat change. Partitioning of the genetic variation into between and within the population as well as between and within the regions of origin revealed that 75% and 88% of the variation was found within the populations and within the regions, respectively. Cluster analysis of genetic distance estimates further confirmed low level of differentiation of wild sorghum populations both on population and regional bases. The implications of the results for genetic conservation purposes are discussed.

Phylogeny in the genus Hordeum based on nucleotide sequences closely linked to the vrs1 locus (row number of spikelets).

The phylogenetic relationship between four basic genomes designated H, I, Xa, and Xu in the genus Hordeum was studied using a nuclear DNA sequence. The sequence, cMWG699, is single copy in the H. vulgare genome, and tightly linked to the vrs1 locus which controls two- and six-rowed spikes. DNA fragments homologous to cMWG699 were amplified from diploid Hordeum species and the nucleotide sequences were determined. A phylogeny based on both base substitutions and an insertion-deletion event showed that the H- and Xa-genome groups are positioned in one monophyletic group indicating that the Xa-genome taxa should be included in the H-genome group. The large H-genome group is highly homogeneous. The I and Xu genomes are distinctly separated from H and Xa, and form sister groups. Another phylogeny pattern based on data excluding the insertion-deletion gave a result that the Xa genome forms a sister group to the H-genome group. The difference between the H and Xa genomes was affected only by a single base insertion-deletion event, thus the H and Xa genomes are likely to be closely related. The I and Xu genomes were again distinctly separated from the H and Xa genomes.

Rat stomach ECL-cell histidine decarboxylase activity is suppressed by ergocalciferol but unaffected by parathyroid hormone and calcitonin.

The ECL cells are peptide hormone-producing cells, rich in histamine and chromogranin A (CGA)-derived peptides, that operate under the control of gastrin. Gastrin and the ECL cells form a functional unit, the gastrin-ECL-cell axis. The aims of the present study were to examine (1) if calcitonin (CT), parathyroid hormone (PTH) and vitamin D affect the gastrin-ECL-cell axis (by measuring the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), and the expression of HDC mRNA and CGA mRNA in the ECL cells), and (2) if activation of the gastrin-ECL-cell axis affects the parathyroid glands (by measuring plasma PTH and mRNA expression). We also examined the possibility that the oxyntic mucosa harbours vitamin D receptors. Fasted rats received intravenous infusion of PTH and CT with or without gastrin. PTH raised the blood Ca2+ concentration, whereas CT infusion lowered it. Plasma PTH rose in response to CT, while serum gastrin remained unaffected. ECL-cell HDC was activated by gastrin but not by CT and PTH. Five daily subcutaneous injections of large amounts of ergocalciferol raised the blood Ca2+ concentration, while reducing the oxyntic mucosal HDC activity and the expression of HDC and CGA mRNA. The serum gastrin concentration was not affected. The findings are in line with the idea that the gastrin-ECL-cell axis can be suppressed by vitamin D or by vitamin D-dependent mechanisms. Western blot analysis revealed the presence of vitamin D receptor immunoreactivity and reverse transcription PCR detected vitamin D receptor gene expression in the rat oxyntic mucosa. Hypergastrinemia was induced by daily peroral treatment with the H+/K+-ATPase inhibitor, omeprazole, for 2 weeks or by continuous subcutaneous infusion of gastrin for 7 days. Elevated serum gastrin concentration was associated with increased HDC activity and increased HDC and CGA mRNA expression in the oxyntic mucosa. There was no elevation of plasma PTH or PTH mRNA expression in the parathyroid gland.

Genome-specific repetitive DNA and RAPD markers for genome identification in Elymus and Hordelymus.

We have developed RFLP and RAPD markers specific for the genomes involved in the evolution of Elymus species, i.e., the St, Y, H, P, and W genomes. Two P genome specific repetitive DNA sequences, pAgc1 (350 bp) and pAgc30 (458 bp), and three W genome specific sequences, pAuv3 (221 bp), pAuv7 (200 bp), and pAuv13 (207 bp), were isolated from the genomes of Agropyron cristatum and Australopyrum velutinum, respectively. Attempts to find Y genome specific sequences were not successful. Primary-structure analysis demonstrated that pAgc1 (P genome) and pAgc30 (P genome) share 81% similarity over a 227-bp stretch. The three W genome specific sequences were also highly homologous. Sequence comparison analysis revealed no homology to sequences in the EMBL-GenBank databases. Three to four genome-specific RAPD markers were found for each of the five genomes. Genome-specific bands were cloned and demonstrated to be mainly low-copy sequences present in various Triticeae species. The RFLP and RAPD markers obtained, together with the previously described H and St genome specific clones pHch2 and pP1Taq2.5 and the Ns genome specific RAPD markers were used to investigate the genomic composition of a few Elymus species and Hordelymus europaeus, whose genome formulas were unknown. Our results demonstrate that only three of eight Elymus species examined (the tetraploid species Elymus grandis and the hexaploid species Elymus caesifolius and Elymus borianus) really belong to Elymus.

Relationships of wild Brassica species with chromosome number 2n = 18, based on RFLP studies.

An array of 10 wild Brassica species with chromosome number 2n = 18 represented by 34 populations was analyzed for genome similarity using genomic and cDNA clones. Species studied included B. bourgeaui (Webb) O. Kuntze, B. cretica Lam., B. hilarionis G.E. Post, B. incana Ten., B. insularis Moris, B. macrocarpa (Guss.) Caruel, B. montana Pourret, B. oleracea L., B. rupestris Rafin., and B. villosa Biv. The RFLP data were used to calculate similarities between populations that were subsequently treated in a cluster analysis. Most populations of a species were grouped together and were separate from populations of other species. The previously identified B. incana - B. rupestris - B. villosa complex was verified, and genetic similarity between the species B. montana and B. oleracea was evident. An interesting association between B. insularis and B. macrocarpa was observed. The UPGMA analysis showed that the species tended to cluster according to geographic region: B. cretica and B. hilarionis comprise a cluster that could be called Eastern Mediterranean; B. oleracea, B. bourgeaui, and B. montana define an Atlantic - Western Mediterranean cluster; B. incana, B. rupestris, and B. villosa form an Italian group; and a B. insularis - B. macrocarpa association may be called Central Mediterranean.

A study of 28 Elymus species using repetitive DNA sequences.

Four repetitive DNA sequences cloned from the barley (Hordeum vulgare) genome and common for different Triticeae species were used for a molecular study of phylogenetic relationships among 28 Elymus species. Two wild Hordeum species (H genome), two Pseudoroegneria species (S genome), Agropyron cristatum (P genome), and Australopyrum velutinum (W genome) were included as genomic representatives for the genomes that supposedly were involved in the evolution of the genus Elymus. Our results are essentially congruent with the genomic classification system. This study demonstrates that Elymus is not a monophyletic genus. Based on an analysis of Southern blot hybridization we could discriminate between SY and SH species owing to the strong specific hybridization pattern of the H genome. Hexaploid SYH species gave a hybridization pattern similar to SH species for the same reason. The results support the genomic composition of Elymus batalinii as SYP and also indicated the presence of at least one H genome in Elymus enysii with a hitherto unknown genomic constitution. Elymus erianthus had a hybridization pattern distinctly different from all other species in the investigation. Key words : Elymus, RFLP, phylogeny, repetitive DNA.

Genetic validity of RAPD markers at the intra- and inter-specific level in wild Brassica species with n=9.

The sequence homology of co-migrating RAPD markers within a genus, across species, and among populations of a species was investigated. DNA was isolated from ten wild Brassica species with n=9 and the RAPD patterns were established using three random primers. Five RAPD markers which appeared to be characteristic for the n=9 species (genus level), four markers which appeared to be species specific, and one population-specific marker were isolated from agarose gels and hybridized to the RAPD profiles of the ten Brassica species. Two RAPD markers were cloned for comparison with gel-isolated RAPD fragment probes in hybridization experiments. Non-specific and background hybridization, occurring when gel-isolated fragments were used as probes, disappeared when cloned fragments were used. A total of 250 RAPD-marker hybridizations were scored according to visual presence or absence in a gel lane. All except three markers hybridized as expected, resulting in an error rate of 1.2%. The deviating results included a lack of hybridization although a band was visible in the gel, a length polymorphism for one marker, and a dual hybridization signal for two single-band markers.

Purification, characterization, and molecular cloning of basic PR-1-type pathogenesis-related proteins from barley.

Partial amino acid sequences of two proteins, purified from barley leaves reacting hypersensitively to the powdery mildew fungus, showed a high degree of amino acid identity to the PR-1 proteins originally described in tobacco. The proteins, subsequently designated HvPR-1a and HvPR-1b, show apparent pI values of approximately 10.5 and 11, respectively and apparent M(r) 15,000. Independently, differential screening of a cDNA library prepared from barley leaves, exhibiting a compatible interaction with the powdery mildew fungus, resulted in isolation of cDNA species representing two PR-1 homologs. With the exception of one amino acid, the partial amino acid sequences of HvPR-1a and HvPR-1b are identical to internal sequences of the polypeptides derived from the two cDNA species. These derived polypeptides are each 164 amino acids long and both have putative N-terminal leader sequences of 24 amino acids. That these proposed leader sequences are functional is indicated by the observed occurrence of both proteins in the intercellular fluid. The proposed mature proteins (calculated M(r) 14,490 and 15,204) share 91% identical amino acids with each other and 56 to 74% with other PR-1 proteins. Northern blot hybridization and immunoblotting, respectively, show that both transcripts and both proteins accumulate following inoculation of susceptible and hypersensitivity resistant barley leaves with the powdery mildew fungus.

Characterization of a family of tandemly repeated DNA sequences in Triticeae.

The recombinant plasmid dpTa1 has an insert of relic wheat DNA that represents a family of tandemly organized DNA sequences with a monomeric length of approximately 340 bp. This insert was used to investigate the structural organization of this element in the genomes of 58 species within the tribe Triticeae and in 7 species representing other tribes of the Poaceae. The main characteristic of the genomic organization of dpTa1 is a classical ladder-type pattern which is typical for tandemly organized sequences. The dpTa1 sequence is present in all of the genomes of the Triticeae species examined and in 1 species from a closely related tribe (Bromus inermis, Bromeae). DNA from Hordelymus europaeus (Triticeae) did not hybridize under the standard conditions used in this study. Prolonged exposure was necessary to obtain a weak signal. Our data suggest that the dpTa1 family is quite old in evolutionary terms, probably more ancient than the tribe Triticeae. The dpTa1 sequence is more abundant in the D-genome of wheat than in other genomes in Triticeae. DNA from several species also have bands in addition to the tandem repeats. The dpTa1 sequence contains short direct and inverted subrepeats and is homologous to a tandemly repeated DNA sequence from Hordeum chilense.

Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences.

A set of six cloned barley (Hordeum vulgare) repetitive DNA sequences was used for the analysis of phylogenetic relationships among 31 species (46 taxa) of the genus Hordeum, using molecular hybridization techniques. in situ hybridization experiments showed dispersed organization of the sequences over all chromosomes of H. vulgare and the wild barley species H. bulbosum, H. marinum and H. murinum. Southern blot hybridization revealed different levels of polymorphism among barley species and the RFLP data were used to generate a phylogenetic tree for the genus Hordeum. Our data are in a good agreement with the classification system which suggests the division of the genus into four major groups, containing the genomes I, X, Y, and H. However, our investigation also supports previous molecular studies of barley species where the unique position of H. bulbosum has been pointed out. In our experiments, H. bulbosum generally had hybridization patterns different from those of H. vulgare, although both carry the I genome. Based on our results we present a hypothesis concerning the possible origin and phylogeny of the polyploid barley species H. secalinum, H. depressum and the H. brachyantherum complex.

Minicircle variation in Beta mitochondrial DNA.

Mitochondrial DNAs from nine male fertile and eight cytoplasmic male sterile (cms) accessions of wild and cultivated Beta beets were investigated for the presence of low molecular weight DNA molecules. Five different supercoiled DNA molecules were detected, varying in size from 1.33 to 1.63 kb. Southern hybridizations revealed multimeric forms and sequence homologies between the minicircles. The occurrence of the different minicircles among the 17 accessions was investigated by agarose gel electrophoresis and Southern hybridization using minicircle specific probes. The 1.33 and 1.63 kb minicircles were found in most accessions, the other three minicircles were found in one or two of the wild Beta beet accessions. The presence of a low number of small, more or less homologous, minicircles in all investigated plants makes these molecules a general characteristic of Beta mtDNA. No association is found between the presence or absence of specific minicircles and the expression of male sterility. Neither does the distribution of the different minicircles in Beta beets indicate any essential biological role of these minicircles.

Hypertension, age, and smoking related to genotoxic sensitivity in human lymphocytes: a population study.

The levels of N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced unscheduled DNA synthesis (UDS) were determined in resting mononuclear leukocytes from males in a geographically defined population. The mutagen sensitivity, as determined by the NA-AAF method, was associated with the risk indicators of cerebrocardiovascular disease (CCVD) rather than with CCVD itself. Thus, high blood pressure, age, and smoking--some of the most important risk indicators of CCVD--seem to be conditions which predispose an individual's mononuclear blood cells to increased likelihood of accumulating DNA damage. Hypertensives evidenced higher values of NA-AAF-induced UDS than did nonhypertensive controls, even though the former were treated in accordance with internationally accepted norms. We have previously shown that hypertensives with normalized blood pressure did not differ from controls, when assessed by the NA-AAF method. In the present study, the hypertensives' blood pressure values were 19/12 mm Hg higher than those of the controls, indicating a need for more intensive treatment. Borderline hypertensives did not differ from their controls when assessed by the NA-AAF method, even though their blood pressure values were 19/9 mm Hg higher than those of their controls. This indicates that the group of borderline hypertensives may be heterogeneous, and that some of the patients may be suffering from a condition different from that in those with established hypertension.

Mutagen sensitivity, smoking habits and enzyme induction in healthy middle-aged men.

Unscheduled DNA synthesis (UDS) (excision-repair) of N-acetoxy-2-acetylaminofluorene (NA-AAF) damage to the DNA of human lymphocytes and levels of 3H-labeled NA-AAF bound to the DNA (carcinogen binding) of lymphocytes after 18 hr of culturing were measured in a consecutive subsample of healthy middle-aged males attending a multiphasic health screening program at the Department of Preventive Medicine in Malmö during 3 weeks in November-December 1981, and compared relative to their smoking habits, body weight, serum cholesterol, and gamma-glutamyltransferase levels as well as aryl hydrocarbon hydroxylase inducibility. This study group numbered 66 males and was uniform in sex, age, and investigation time. No case of significant arterial hypertension was present. The UDS and carcinogen binding results showed no correlation with the other factors measured, with the exception of smoking which was strongly (P less than 0.01) associated with increasing levels of both the UDS and carcinogen binding values. It is concluded that under ordinary circumstances smoking may represent the most important exogenous factor which may modulate risk to cardiovascular disease and cancer by influencing individual mutagen sensitivity.

The effects of smoking, hypertension and cardiovascular disease on the estimation of unscheduled DNA synthesis and covalent binding to DNA induced by N-acetoxy-2-acetylaminofluorene in resting human mononuclear leukocytes.

The levels of N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced unscheduled DNA synthesis (UDS) and of NA-AAF binding to DNA have been determined in resting mononuclear leukocytes from individuals with various smoking habits, heart infarct patients and subjects diagnosed for hypertension. Age-matched and blood-pressure-controlled smokers (n = 99) had significantly elevated levels of NA-AAF-induced UDS and NA-AAF binding to DNA when compared to nonsmokers (n = 75) similarly corrected for age and blood pressure. Heart infarct patients without any history of risk factors, as well as diagnosed hypertensives with normalized blood pressure, were not significantly different from matched controls when assessed by the NA-AAF method. Our results support the theory that increased mutagen sensitivity is associated with smoking and high blood pressure but not with cardiovascular disease itself via some mechanism of genetic selection.

A genetic component of the variance of N-acetoxy-2-acetylaminofluorene-induced DNA damage in mononuclear leukocytes determined by a twin study.

The level of N-acetoxy-2-acetylaminofluorene (NA-AAF)-induced unscheduled DNA synthesis and the level of covalent binding of NA-AAF to DNA were determined in the mononuclear leukocytes of monozygotic and diazygotic twin pairs (n = 16 for each type). A statistically significant high degree of heritability was calculated for both parameters which, in turn, indicate genetic control of individual levels of induced DNA damage by NA-AAF.

Occupational and in vitro exposure to styrene assessed by unscheduled DNA synthesis in resting human lymphocytes.

Lymphocytes from 38 individuals occupationally exposed to styrene concentrations in workroom air of 1 p.p.m. t0 40 p.p.m. were examined for any genotoxic effects using unscheduled DNA synthesis (UDS) as the indicator of DNA damage. The mean level of N-acetoxy-2-acetylaminofluorene (NA-AAF) induced UDS was significantly increases (p less than 0.001) for the styrene exposed group when compared to the mean level for the unexposed controls. There was no significant effect on u.v.-induced UDS from the in vivo styrene exposure. Lymphocyte cultures exposed in in vitro to styrene concentrations up to 100 micro M have confirmed the UDS data collected on individuals occupationally exposed to styrene. In addition, the in vitro study has also shown that the increased NA-AAF induced UDS resulting from styrene exposure was paralleled by a similar increase in NA-AAF binding to DNA. Taken together these results indicate that styrene exposure does not inhibit DNA repair synthesis, but rather it predisposes lymphocytes to an increased risk for DNA damage induction from subsequent genotoxic exposures.