We present evidence that two distinct regions of the DNA upstream from the mouse metallothionein-I gene contain metal-responsive regulatory sites. This result was obtained by analyzing a systematic series of deletion, insertion, duplication, and clustered point mutations introduced into cultured cells on a simian virus 40 plasmid vector. The two upstream regions contain a duplicated evolutionarily conserved DNA sequence. While either upstream region is sufficient to confer heavy metal responsiveness, both are required to give maximal levels of induced transcription.The metallothioneins (MTs) are small cysteine-rich proteins that tightly bind heavy metals. They are present in a broad range of eukaryotes, from yeast to man, and are expressed in many different tissues and cell types. Exposure to heavy metals results in a rapid increase in MT mRNA levels and protein synthesis in both cultured cells and whole animals. This homeostatic regulatory mechanism plays a critical role in protecting cells against toxic ions, such as cadmium and mercury, and may also be involved in the metabolism of essential elements such as zinc and copper (reviewed in ref. 1). The metal response, which occurs in the presence of protein synthesis inhibitors, can be attributed largely if not exclusively to increased transcription rates (2-5).What MT gene DNA sequences are involved in the rapid transcriptional response to heavy metals? We have shown previously that a cloned mouse MT-I gene, containing 2000 base pairs (bp) of 5' flanking DNA, retains its ability to be induced by cadmium when introduced into cultured monkey kidney cells on simian virus 40 (SV40) viral and plasmid vectors (4). Appropriate regulation has also been observed for mouse and human MT and MT fusion genes carried on episomal bovine papilloma virus vectors (6, 7), introduced into cultured mouse or rat cells by transformation (5, 8), microinjected into mouse eggs (9), or integrated into the genome in transgenic mice (10). To more precisely localize and characterize the MT regulatory sequences, we have constructed a systematic series of 3' deletions, 5' deletions, internal deletions, duplications, insertions, and clustered point mutations in the mouse MT-I gene. These were introduced into cultured cells on an SV40 plasmid vector and analyzed both for their efficiency of transcription and for their ability to be induced by cadmium. We have also compared the 5' flanking DNA sequences of the mouse MT-I gene with three functional human MT genes. Our results show that the metalresponsive elements lie in at least two distinct regions of the 5' flanking DNA. These regions share a conserved nucleotide sequence that may serve as a recognition signal for cellular regulatory factors. MATERIALS AND METHODSRecombinant plasmids were constructed and characterized by standard methods (11). The starting pML-SV40-MT construct, JYMMT(E), was described previously (4). The Escherichia coli galactokinase-SV40 transcription unit was obtained from pSVK105A, a derivative of pSVK carrying a ...

We have estimated the potential phylogenetic utility of the ribosomal external transcribed spacer (ETS) from the nuclear ribosomal region. The ETS was sequenced from 13 annual Medicago (Fabaceae) species upstream a highly conserved motive which was found among many different organisms. In the genus Medicago, the ETS was found to evolve 1.5 times faster than the internal transcribed spacer and to be 1.5 times more informative. Reduced ribosomal maturation process constraints on ETS are proposed to explain the different evolutionary rates between the two spacers. Maximal phylogenetic resolution and support was obtained when the two spacers were analyzed together. No incongruence between the two spacers was found and ETS appears to be a valuable source of information for solidifying ITS plant phylogeny. The phylogeny obtained in Medicago suggests that none of the three subsections included in the study is monophyletic.

Sequences of the internal transcribed spacers (ITSs) of 18S-26S nuclear ribosomal DNA were used to resolve phylogenetic relationships and chromosomal evolution among 14 species of the genus Hypochaeris (Asteraceae). Parsimony analysis was performed for phylogenetic reconstruction, and sequence divergence between species was estimated. Pairwise sequence divergence within Hypochaeris genus ranged from 0% to 25.68% in ITS1 and from 0% to 17.08% in ITS2. A highly resolved strict-consensus tree was obtained that showed the phylogenetically useful information of ITS sequences within the genus Hypochaeris. Four clades could be well distinguished, one of them formed by the single species H. robertia, which appeared to be the most related to the ancestral species of the genus. The results agree with taxonomic classification based on morphological data, and the tree obtained, when indels are coded as missing data, aggregates the species having the same chromosome number, except in one clade. According to the ITS phylogenetic tree, the chromosomal evolution within the genus Hypochaeris conflicts with the previous hypothesis and suggests that karyotype evolution in Hypochaeris was accompanied with both decreasing and increasing dysploidy, probably with several chromosomal rearrangements, and from an ancestral basic chromosome number of 4 or 5.

Summary Boisselier‐Dubayle, M. C., Jubier, M. F., Lejeune, B. & Bischler, H.: Genetic variability in the three subspecies of Marchantia polymorpha (Hepaticae): isozymes, RFLP and RAPD markers. – Taxon 44: 363‐376. 1995. – ISSN 0040‐0262. Three subspecies of Marchantia polymorpha have been recently characterized by isozyme patterns, ecology and morphological characters. To confirm their taxonomic status and to better understand their relationships and genetic variability, a previous study on enzyme polymorphism was extended to additional colonies and enzymes. A parallel study was done with DNA markers derived from restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Results obtained with these different approaches (protein and DNA level) all support the taxonomic distinctness of the subspecies. Genetic similarity within subspecies was found to be high over wide geographic areas, and low among subspecies. The genetic divergence of the three subspecies may be taken to illustrate a case of speciation with adaptation to different ecological niches, and subsequent reproductive isolation. The hybrid origin of Marchantia polymorpha subsp. ruderalis, postulated in the past, is not supported by our data.

The aim of this study was to find molecular markers (RAPD and SCAR) for the wheat leaf rust resistance gene Lr24. A backcross line, RL 6064, possessing a single resistance gene to leaf rust (Lr24) and its recurrent parent 'Thatcher' were used to find RAPD markers linked to the Lr24 gene. Among 125 RAPD primers tested, only one (OP-H5) detected an additional band in the resistant line RL 6064. The genetic linkage of this molecular marker to Lr24 was tested on a segregating F2 population derived from a cross between the leaf rust resistant line RL 6064 and the susceptible line 'Chinese Spring'. This marker showed complete linkage to the Lr24 resistance gene. A more reliable and specific marker for this resistance gene was made by converting it into a sequence characterized amplified region (SCAR). The presence of a single amplification product allowed direct detection of the gene in the test tube by the addition of ethidium bromide. This SCAR marker linked to the leaf rust resistance gene Lr24 could be used easily in a practical breeding program.

The mitochondrial single-copy gene nad5 of wheat and maize consists of 5 exons located on three widely separated regions of the genome that are independently transcribed. The first region contains exons I and II separated by an atypical group II intron; in the second region is exon III (only 22 bp long), which is flanked upstream by a maturaserelated open reading frame (ORF) and exon e of the nadl gene, and downstream by a previously unidentified ORF (ORF143); in the third region are exons IV and V separated by a group II intron. In maize, this last domain is flanked upstream by the genes rps72, nadd, and tRNASer and downstream by a chloroplast tRNACys. RNA editing occurs in wheat exons IV and V as C -t o 4 changes. A detailed analysis of the transcription of the nad5 gene in wheat and maize reveals that the exons are assembled into a 2.4-kb mRNA after two cis-splicing (between exons I and II and exons IV and V) and two trans-splicing events. The trans-splicing process involves the sequences flanking exons II, 111, and IV that feature group II introns. A model is proposed for the assembly and maturation of the nad5 transcripts.

The Holcus complex in France consists of two species, Holcus lanatus L. (Yorkshire fog; 2n= 2x= 14) and Holcus mollis L. (Creeping soft‐grass; 2n= 4x= 28) and an interspecific hybrid H.m.×H.l. (2n= 5x= 35), which is morphologically similar to Holcus mollis. A heterologous rDNA probe from wheat was used to detect the corresponding region in Holcus (s. l.) genomic DNA'fragments, for six to eight plants from 13 populations located south‐west of Paris. A restriction enzyme map of the ribosomal RNA genes (rDNA) in Holcus (s. l.) was also constructed. The length polymorphism detected in the IGS region was used as a DNA fingerprint for the identification of different cytotypes and species of the Holcus complex and for the typing and delimitation of individuals in populations. In the light of the results we reconsider the assumption that the pentaploid hybrid H.m.×H.l. is purely clonal. New hypotheses concerning the origin of the pentaploid hybrid and its reproduction are proposed, and the consequences for genetic diversity in natural populations discussed.

The mitochondrial genome of the selfed progeny of a plant regenerated from long-term somatic tissue culture displays specific structural rearrangements characterized by the appearance of novel restriction fragments. A mitochondrial DNA library was constructed from this selfed progeny in the SalI site of cosmid pHC79 and the novel fragments were subsequently studied. They were shown to arise from reciprocal recombination events involving DNA sequences present in the parental plant. The regions of recombination were sequenced and the nucleotide sequences were aligned with those of the presumptive parental fragments. We characterized an imperfect short repeated DNA sequence, 242 bp long, within which a 7-bp DNA repeat could act as a region of recombination. The use of PCR technology allowed us to show that these fragments were present in both parental plants and tissue cultures as low-abundance sequence arrangements.