AFLP maps of Petunia hybrida: building maps when markers cluster.

AFLP mapping in Petunia hybrida was undertaken with the intention of building a high-density genetic map suitable for applications such as map-based gene cloning. In total five maps were constructed from two mapping populations, with placement of more than 800 markers. Despite the large number of markers the resulting map is roughly ten-fold smaller than those of other plant species, including the closely related tomato. Low levels of recombination are reflected in clusters of tightly linked markers, both AFLPs and RFLPs, in all the maps. Clustering patterns vary between mapping populations, however, such that loci tightly linked in one population may be separable in another. Combined with earlier reports of aberrant meiotic pairing and recombination, our results suggest that, for species like petunia, map-based cloning may be more complex than in model species such as arabidopsis and tomato.

Towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a Mannheimia haemolytica A1 leukotoxin 50 fusion protein.

Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly on Mannheimia haemolytica A1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated. Derivatives of the M. haemolytica Lkt in which the hydrophobic transmembrane domains were removed were made. Lkt66 retained its immunogenicity and was capable of eliciting an antibody response in rabbits that recognized and neutralized authentic Lkt. Genes encoding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation. Constructs were screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco. The fusion construct pBlkt50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for transcription, was selected and introduced into white clover by Agrobacterium tumefaciens-mediated transformation. Transgenic lines of white clover were recovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. Lkt50-GFP was found to be stable in clover tissue after drying of the plant material at room temperature for 4 days. An extract containing Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt. This is the first demonstration of the expression of an M. haemolytica antigen in plants and paves the way for the development of transgenic plants expressing M. haemolytica antigens as an edible vaccine against bovine pneumonic pasteurellosis.

Anthocyanin regulatory mutations in pea: effects on gene expression and complementation by R-like genes of maize.

Anthocyanin production in higher plants is a function of the tissue considered and its developmental stage, and is modulated by environmental factors. In maize, the best characterized system, regulation of the pathway is achieved largely through the action of proteins with homology to the transcriptional factors encoded by myc and myb proto-oncogenes of animals; these homologues control the expression of structural genes and thus regulate the availability of anthocyanin biosynthetic enzymes. We have studied anthocyanin biosynthesis and its regulation in flowers of pea (Pisum sativum). Our results demonstrate a correlation between anthocyanin accumulation and steady-state mRNA levels for genes encoding chalcone synthase, flavanone 3 beta-hydroxylase, and dihydroflavonol 4-reductase in developing flowers. Patterns of expression for these biosynthetic genes in both a and a2 mutants confirm the regulatory roles of the two a loci. The reduced expression of all three biosynthetic genes in mutant lines suggests that genes acting both early and late in the anthocyanin biosynthetic pathway are controlled by a and a2. Particle bombardment of floral tissue demonstrates the ability of two maize R-like genes, Lc and R-S, but neither myb-like genes nor R-like genes from snapdragon or petunia, functionally to complement a and a2 mutations. We cannot distinguish whether a and a2 act coordinately or sequentially in anthocyanin regulation, but the epistatic action of maize R-like genes suggests that they mimic the action of a gene that normally functions downstream of both a and a2 in the regulatory cascade.

Myb26: a MYB-like protein of pea flowers with affinity for promoters of phenylpropanoid genes.

Plant Myb proteins represent a group of transcription factors which have a DNA-binding domain similar to that found in the products of the animal myb proto-oncogenes. Members of the Myb family regulate the biosynthesis of phenylpropanoids, including anthocyanin and phlobaphene pigments, in several species. In this study, PCR with degenerate primers was used to analyse the presence of myb-like genes in pea (Pisum sativum L.). A fragment representing a flower bud-expressed gene, designated myb26, was recovered, and a full length cDNA was isolated from a pea flower bud cDNA library. The predicted protein is 217 amino acids long and its Myb-domain and carboxy terminal region show extensive homology to the snap-dragon proteins Myb305 and Myb340, both of which regulate phenylpropanoid biosynthesis. Expression of myb26 is flower-specific and parallels the expression of flavonoid biosynthetic genes, increasing as the flower bud matures and intensifies in colour. However, myb26 represents neither of the two known regulatory genes for anthocyanin production in pea, nor does it complement mutants of the myb-like anthocyanin regulatory gene an2 in petunia. Myb26 was expressed in E. coli as a fusion protein. It was shown that in vitro Myb26 recognizes the c-Myb and P-box-like binding sites representing cis-elements in the promoter regions of several phenylpropanoid biosynthetic genes. The results suggest that myb26 is a previously undefined gene involved in regulation of some aspect of phenylpropanoid production in pea.

Isolation and characterization of the Adh2 5' region from Petunia hybrida.

The Adh2 gene from Petunia hybrida has been difficult to clone; exons 1 to 8 were isolated using PCR after unsuccessful screening of three genomic libraries. A combination of inverse and direct PCR strategies has been used to isolate upstream regions of Adh2. Here we report the cloning strategy for the nucleotide sequence of the 5' region of Adh2 from P. hybrida, the locations of the transcriptional start site and putative TATA box, as well as comparative analyses of the upstream regions of petunia Adh2, other Adh genes and other genes induced by hypoxia.

Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

Structure, expression, chromosomal location and product of the gene encoding ADH1 in Petunia.

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated from Petunia hybrida cv. V30 by screening a Petunia genomic library with a maize Adh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of two Adh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay in Petunia protoplasts. We have designated this gene Petunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction of Adh1 mRNA.

The Mu1 maize transposable element induces tissue-specific aberrant splicing and polyadenylation in two Adh1 mutants.

Insertions of the maize transposable element Robertson's Mutator (Mu) into intron 1 of the Adh1 gene have produced a number of mutant alleles altered in quantitative expression. It has previously been shown that transcription and mRNA accumulation are reduced for two of these alleles, Adh1-S3034 and Adh1-S4477. In this report, we describe the presence of Mu1-hybridizing polyadenylated transcripts in roots of anaerobically induced seedlings of these same mutants. Sequence analysis of Mu1-hybridizing clones from a cDNA library of S3034 RNA indicated that these transcripts originated from the Adh1 locus and were produced by alternative processing of S3034 pre-mRNA. Approximately half of the cDNAs represented transcripts that had not undergone excision of the intron containing the 1.4-kilobase Mu1 insertion but were processed in response to signals present in the transposable element. Mu1 contains a donor splice site in the 5'-terminal inverted repeat that can be joined to the Adh1 exon 2 acceptor, resulting in removal of most of the Mu1 sequences from the pre-mRNA; alternatively this donor can be spliced to an acceptor within Mu1, removing an 89-nucleotide intron. Mu1 also contains polyadenylation signals that are used to produce truncated transcripts. These Mu1 transcripts produced by aberrant splicing and polyadenylation were not detected in RNA isolated from developing kernels.

The effect of insertion of the maize transposable element mutator is dependent on genetic background.

A secondary mutant, derived from an allele of maize alcohol dehydrogenase 1 (Adh1) carrying a Mutator transposable element (Mu1) in its first intron, was reported to exhibit a threefold decrease in ADH enzymatic activity and steady-state RNA levels compared to the original mutant. The original mutant, Adh1-S3034 (abbreviated S3034), was previously characterized at the molecular level. The derivative, abbreviated S3034b, has now been cloned; at the DNA sequence level the insertion and surrounding Adh1 sequences are indistinguishable from S3034. Furthermore, in our lines there is no difference in relative ADH activities between products of the two putative alleles. A comparison of gene expression in heterozygotes obtained by crossing to different tester lines reveals a correlation between the measured decrease in levels of ADH polypeptide produced by the mutant allele and the background in which it is measured; this effect is distinct from any background-related variation in the expression of the progenitor allele. It does not appear to be attributable to alternative patterns of DNA modification. It appears to reflect a background-associated difference in the level of normal Adh1-RNA produced. Thus the previously reported distinction between S3034 and S3034b may be due to differences in the extent to which the mutant allele and a given genetic background interact to produce functional Adh1-RNA.

Chromosome breakage undetectable in active Mu lines of maize.

We have used a set of Mutator-induced mutants of Bz1 to test whether members of the Mutator (Mu) family of maize transposable elements produce broken chromosomes. From our inability to demonstrate the simultaneous loss of two dominant endosperm markers distal to Mu insertions at Bz1 we conclude that either Mu, unlike many elements of the Ds family, does not induce such breaks, or it does so at a very low frequency.

Mu1-induced mutant alleles of maize exhibit background-dependent changes in expression and RNA processing.

We have examined effects of mutations created by transposition of the Mu1 element of maize into genes coding for Adh 1 and Sh 1, by means of allozyme measurements, DNA and RNA hybridization, cloning, and sequencing. From our analysis of mutant alleles we conclude that the element acts both to reduce steady-state levels of RNA and to induce aberrant processing of primary transcripts. We also conclude that genetic background can exert considerable influence in determining the degree to which Mu1 affects these aspects of gene expression.

Insertion of Mu into the Shrunken 1 gene of maize affects transcriptional and post-transcriptional regulation of Sh1 RNA.

Insertion of a Mu transposable element at the Shrunken 1 (Sh1) locus of maize has resulted in kernels with the typical collapsed appearance of sh mutants. Molecular analysis of the mutant gene has revealed the presence of a 1.4 kb insertion immediately upstream from the normal transcriptional start site. Mu insertion has brought about a series of changes in gene expression: the mRNA cap site has been shifted downstream so that it now lies inside the Mu element; transcription is reduced approximately sixfold, and the sh mRNA steady-state level is less than 4% of that found in the nonmutant. This disparity reflects a mutational defect in post-transcriptional regulation which is manifested as a decrease in Sh RNA abundance.

Anaerobic treatment of maize roots affects transcription of Adh1 and transcript stability.

Among the adaptations to stress exhibited by plants is the anaerobic response of roots, induced by submerging roots in water. The response consists of a programmed change in gene expression: proteins produced under aerobic conditions are no longer synthesized but are replaced by approximately 20 so-called anaerobic peptides (M. M. Sachs, M. Freeling, and R. Okimoto, Cell 20:761-767, 1980). The gene for maize alcohol dehydrogenase I (Adh1) is expressed at high levels under such conditions. We report here that changes in alcohol dehydrogenase I RNA levels in anaerobic roots are associated with changes in both transcription rate and transcript stability.

Insertion of an unstable element in an intervening sequence of maize Adh1 affects transcription but not processing.

Three independently isolated and unstable mutants of the maize alcohol dehydrogenase 1 gene (Adh1) have arisen by insertion of the Mu transposing element into the first intervening sequence of the progenitor Adh1 allele. The mutants have been selected for their decreased levels of alcohol dehydrogenase 1 activity. The original mutants were unstable, giving rise to both revertant alleles and altered mutant alleles. From one of the original mutants, two derivative mutants have been recovered and described. We analyzed the effect of Mu insertion in all five of these mutants by measuring relative levels of run-off transcripts from the progenitor and mutant alleles and by comparing levels of run-off transcripts corresponding to regions lying 5' and 3' to the insertion sites. In this paper we present evidence that early transcriptional events are affected, but that, in spite of the inclusion of a 1.4-kilobase transposing DNA element, processing of transcripts occurs normally.

Exceptionally High Levels of Restriction Site Polymorphism in DNA near the Maize Adh1 Gene.

Restriction maps have been prepared for the chromosomal region near seven biochemically and genetically distinct maize alcohol dehydrogenase-1 (Adh1) alleles using a small cDNA probe for Adh1. Five restriction sites spanning about 4 kb in and near the Adh1 transcription unit appear identical in all seven alleles. Outside this conserved region, variation in restriction site position is the rule. Six of the seven alleles are distinguishable, and the alleles appear to fall into four groups. The DNA flanking the 1S-type alleles seems to share no restriction site homology with the DNA near the 1F-type alleles. Several hypotheses are put forward to explain how such high levels of polymorphism could have arisen in a species that has been domesticated for only about 10,000 years.

Cellular sequences are present in the presumptive avian myeloblastosis virus genome.

EcoRI restriction endonuclease fragments from a lambda proviral DNA hybrid containing the entire presumptive avian myeloblastosis virus (AMV) provirus, and from a lambda proviral hybrid containing a partial myeloblastosis-associated virus type 1 (MAV-1)-like provirus were compared by heteroduplex analysis. The cloned presumptive AMV provirus was also analyzed by electron microscopy, using R-loop formation with purified 35S RNA isolated from virions of the standard AMV complex. The results indicate that the putative AMV genome contains a segment absent in its MAV-1-like helper virus. This segment represents a substitution in the region of the genome that in MAV-1 virus is occupied by the envelope gene and is approximately 900 +/- 160 nucleotide pairs in length. Hybridization of specific probes from the presumptive AMV genome to Southern blots of EcoRI-digested cellular DNA has revealed that these substituted sequences are homologous to chicken and duck DNA that is not related to chicken endogenous proviral sequences.

Isolation of the chicken beta-globin gene and a linked embryonic beta-like globin gene from a chicken DNA recombinant library.

A library of random chicken DNA fragments, 15-22 kb long, has been prepared in the vector lambda Charon 4A. This library was screened with combined adult and embryonic globin cDNA, and several independent globin gene-containing recombinants were isolated. One of these recombinants, lambda Chicken beta-globin 1 (lambda C beta G1), contains the adult chicken beta-globin gene and a closely linked embryonic beta-like globin gene. Both genes are transcribed in the same direction with the adult gene located 5' to the embryonic gene. Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to lambda C beta G1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian beta-globin genes. The adult beta-globin gene also contains a minor (approximately 100 bp long) intervening sequence analogous to the one observed in mammalian beta-globin genes. Restriction enzyme analysis of the adult beta-globin gene on lambda C beta G1 is consistent with the hypothesis that its two intervening sequences occur in the same positions with respect to the beta-globin amino acid sequence as do the corresponding mammalian intervening sequences.

Recombinant DNA clones constructed from immunoglobulin kappa light chain messenger RNA.

Recombinant DNA clones have been generated from mouse myeloma MOPC 21 immunoglobulin kappa light chain mRNA. Complementary DNA (cDNA) synthesized on kappa light chain mRNA by reverse transcriptase was made double stranded and inserted into the bacterial plasmid vector, pMB9. Approximately 70 tetracycline-resistant transformed colonies containing kappa light chain mRNA sequences were identified by colony hybridization. Five of these recombinant clones were selected and characterized. Three clones contain both kappa light chain constant and variable region sequences. Two of these three recombinant clones have been shown to include all of the kappa light chain constant and variable region coding sequences. Another of the five selected recombinant clones contain kappa light chain constant region sequences. The remaining characterized clone appears to be derived from sequences at the 5'-end of kappa light chain mRNA, possibly extending to the terminal cap structure.