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In most crop species, primary productivity depends mainly on the leaf. However, the genes that contribute to the making of plant leaves remain largely unknown. With a view to identifying the genes involved in leaf development in Arabidopsis thaliana, we previously isolated EMS-induced mutants with abnormally shaped leaves and demonstrated that they fall into 94 complementation groups. We present here the map positions of 76 of these genes, which have been obtained using a high-throughput genetic mapping method, based on the simultaneous coamplification by PCR of 21 polymorphic microsatellites and the semiautomated fluorescent detection of the products. The map positions and F2 mapping populations obtained in this work will be instrumental in the positional cloning of these genes, which are essential for leaf development.

In an attempt to identify genes involved in the control of leaf morphogenesis, we have studied 13 Arabidopsis thaliana mutants with curled, involute leaves, a phenotype herein referred to as Incurvata (Icu), which were isolated by G. Röbbelen and belong to the Arabidopsis Information Service Form Mutants collection. The Icu phenotype was inherited as a single recessive trait in 10 mutants, with semidominance in 2 mutants and with complete dominance in the remaining 1. Complementation analyses indicated that the studied mutations correspond to five genes, representative alleles of which were mapped relative to polymorphic microsatellites. Although most double-mutant combinations displayed additivity of the Icu phenotypes, those of icu1 icu2 and icu3 icu4 double mutants were interpreted as synergistic, which suggests that the five genes studied represent three independent genetic operations that are at work for the leaf to acquire its final form at full expansion. We have shown that icu1 mutations are alleles of the Polycomb group gene CURLY LEAF (CLF) and that the leaf phenotype of the icu2 mutant is suppressed in an agamous background, as is known for clf mutants. In addition, we have tested by means of multiplex RT-PCR the transcription of several floral genes in Icu leaves. Ectopic expression of AGAMOUS and APETALA3 was observed in clf and icu2, but not in icu3, icu4, and icu5 mutants. Taken together, these results suggest that CLF and ICU2 play related roles, the latter being a candidate to belong to the Polycomb group of regulatory genes. We propose that, as flowers evolved, a new major class of genes, including CLF and ICU2, may have been recruited to prevent the expression of floral homeotic genes in the leaves.

A non-radioactive, rapid and sensitive method is presented for the simultaneous detection of several mRNA molecules. The technique is based on conventional first-strand cDNA synthesis by reverse transcriptase, followed by multiplex polymerase chain reaction (PCR) co-amplification of several gene products in a reaction mix containing several primer sets, each including a fluorescently labeled oligonucleotide. The PCR products obtained are finally electrophoresed in a single lane of a polyacrylamide gel, in an automated DNA sequencer controlled by fragment-analysis software. The method has proven useful to efficiently detect nine mRNA transcripts, some of which are low copy number, from small specimens such as single flowers and leaves of Arabidopsis thaliana (L.) Heynh. This approach might be easily extended to other biological systems, for developmental and physiological analyses, population studies and diagnosis.

Stress caused by the increased salinity of irrigated fields impairs plant growth and is one of the major constraints that limits crop productivity in many important agricultural areas. As a contribution to solving such agronomic problems, we have carried out a large-scale screening for Arabidopsis thaliana mutants induced on different genetic backgrounds by EMS treatment, fast neutron bombardment, or T-DNA insertions. From the 675,500 seeds we screened, 17 mutant lines were isolated, all but one of which yielded 25-70% germination levels on 250 mm NaCl medium, a condition in which their ancestor ecotypes are unable to germinate. Monogenic recessive inheritance of NaCl-tolerant germination was displayed with incomplete penetrance by all the selected mutants, which fell into five complementation groups. These were named SALOBRENO (SAN) and mapped relative to polymorphic microsatellites, the map positions of three of them suggesting that they are novel genes. Strains carrying mutations in the SAN1-SAN4 genes display similar responses to both ionic effects and osmotic pressure, their germination being NaCl and mannitol tolerant but KCl and Na(2)SO(4) sensitive. In addition, NaCl-, KCl-, and mannitol-tolerant as well as abscisic-acid-insensitive germination was displayed by sañ5, whose genetic and molecular characterization indicates that it carries an extremely hypomorphic or null allele of the ABI4 gene, its deduced protein product lacking the APETALA2 DNA binding domain.

In contrast to bacterial, fungal and vertebrate ornithine transcarbamylases (OTCs; EC 2.1.3.3), very little is known about the enzyme in plants. We report here the isolation of a T-DNA-tagged mutant displaying sensitivity to ornithine, whose characterization has allowed for the identification of several complementary and genomic DNA clones encoding the OTC and auxilin-like 1 (AUL1) proteins of the crucifer Arabidopsis thaliana. Transcript mapping revealed that at least 22 bp within the OTC-AUL1 intercoding region are transcribed from both strands, which makes this one of the rarely described cases of convergent and overlapping transcription units in the nuclear genome of a multicellular eukaryote. Transcription of the OTC gene was shown to be ubiquitous in aerial organs of adult plants, whereas that of AUL1 was obscured by the existence of a putative second copy of the gene. The OTC-AUL1 locus maps at the bottom of chromosome 1.

As a contribution to a better understanding of the developmental processes that are specific to plants, we have begun a genetic analysis of leaf ontogeny in the model system Arabidopsis thaliana by performing a large-scale screening for mutants with abnormal leaves. After screening 46,159 M2 individuals, arising from 5770 M1 parental seeds exposed to EMS, we isolated 1926 M2 putative leaf mutants, 853 of which yielded viable M3 inbred progeny. Mutant phenotypes were transmitted with complete penetrance and small variations in expressivity in 255 lines. Most of them were inherited as recessive monogenic traits, belonging to 94 complementation groups, which suggests that we did not reach saturation of the genome. We discuss the nature of the processes presumably perturbed in the phenotypic classes defined among our mutants.

Branching net-like structures are a trait common to most multicellular organisms. However, our knowledge is still poor when it comes to the genetic operations at work in pattern formation of complex network structures such as the vasculature of plants and animals. In order to initiate a causal analysis of venation pattern formation in dicotyledonous plant leaves, we have first studied its developmental profile in vegetative leaves of a wild-type strain of the model organism Arabidopsis thaliana. As landmarks of the complexity of the venation pattern, we have defined three main developmental parameters, which have been quantitatively followed in time: the ratios of (a) the length and (b) the number of branchpoints of the vein network with the surface of the lamina, which decrease in parallel as the leaf grows, only small differences existing between successive leaves, and (c) the number of hydathodes per leaf, which increases both during leaf expansion and from juvenile to adult rosette leaves. We next searched for natural variations in the first vegetative leaves of 266 ecotypes, finding only 2 which showed a venation pattern unequivocally different from that of the rest, Ba-1 and Ei-5, the latter displaying an extremely simple pattern that we have called Hemivenata. This phenotype, which is inherited as a monogenic recessive trait, is visible both in leaves and in cotyledons and seems to arise from a perturbation in an early acting patterning mechanism. Finally, we have screened for mutants with abnormal venation pattern but normally shaped leaves, concluding that such a phenotype is rare, since only one recessive mutation was obtained, extrahydathodes, characterized by the presence of an increased number of hydathodes per leaf., (Copyright 1999 Academic Press.)

An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T-DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T-DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with NdeI and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T-DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T-DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T-DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T-DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.

The Ultrabithorax (Ubx) gene of Drosophila melanogaster includes two functionally distinguishable regions. One is the Ubx transcription unit, which gives rise by alternative splicing to a family of morphogenetic Ubx proteins (UBX). The other is its upstream bithoraxoid (bxd) region. On the basis of genetic and molecular studies, it is generally assumed that the Ubx transcription unit contains internal positively acting cis-regulatory elements controlling UBX expression in the T3a compartment of the body of Drosophila, while the bxd region contains positive cis-regulatory elements controlling UBX expression in the T3p and A1a compartments. We have performed a genetic analysis of bx bxd cis double mutant chromosomes containing one mutation (bx alleles) affecting the Ubx unit, and a second (bxd alleles) affecting the bxd region of the Ubx gene. Our study of different bx bxd/bx combinations shows that bxd alleles partially rescue the adult mutant phenotypes of bx alleles, which suggests that the bxd region contains a negative cis-regulatory element involved in the control of the activity of the Ubx gene in the T3a compartment.

The Ultrabithorax (Ubx) gene is required during embryogenesis and larval development to specify the third thoracic and first abdominal segments of Drosophila melanogaster. Mutations in the bithoraxoid (bxd) region, a 40 kb DNA stretch upstream of the Ubx promoter, affect cis-regulatory elements responsible for the ectodermal expression of the Ubx gene in the posterior compartment of the third thoracic segment and anterior compartment of the first abdominal segment. Our genetic data and the available molecular information are used to map the adult epidermal cis-regulatory elements within the bxd region. Genetic combinations involving mutations affecting the bxd region show that (1) redundant or cooperatively acting sequences are required for Ubx gene expression in the anterior compartment of the first abdominal segment, and (2) the expression of Ubx in the posterior compartment of the third thoracic segment is modulated by positive and negative cis-regulatory elements.

Two computer packages have been developed to teach bacterial genetics on an introductory genetics course for undergraduate students in biology. The first package, 'CONJUGACION', is designed to teach bacterial conjugation and its genetic outcomes. It includes four main parts. Firstly, a tutorial part presents a theoretical framework using screens of text and animated graphics. Secondly, an interactive concept application section requires students to carry out experiments for the determination of the correct sex and genotype of 10 bacterial strains. The third part uses the previously obtained data for simulating interrupted mating experiments and mapping the bacterial genome. Finally, an evaluation section allows the students to test their understanding through a series of multiple choice questions. The second package, 'LURIDEL', is intended for teaching the preadaptative character of mutation in bacterial populations on the basis of the fluctuation test of Luria and Delbrück. It simulates, graphically, the appearance of mutations in microbial cultures and gives results of simulated fluctuation experiments. Programs were written under the PCOS operating system in MBASIC extended to graphics for running on Olivetti M20 microcomputers.