Allelic variation and differential expression at the 27-kilodalton zein locus in maize.

Allelic variation between inbred lines at the 27-kilodalton zein gene locus in maize has been used to study gene expression in developing endosperm. The inbred lines W22 and W23 contain two genes for this protein within two tandem repeats; the individual genes are virtually identical, with 99.9% homology in the 5'-flanking regions. Using gene-specific oligonucleotide probes, we have shown that transcripts of the downstream gene are found at a 2.5-fold-higher level than those of the upstream gene. Another inbred line, BSSS53, has one copy of the gene which is a recombinant of the duplicated genes at the 3'-flanking region. This line has been used in reciprocal crosses to demonstrate dosage effects for the overexpression of the downstream gene and to show that the overexpression of mRNA is reflected in a corresponding increase in the protein level. The accumulation of the protein through development does not, however, always correspond to the difference in mRNA levels.

Sequence and spatial requirements for the tissue- and species-independent 3'-end processing mechanism of plant mRNA.

Two cis-regulatory regions are required for efficient mRNA 3'-end processing of the maize 27-kDa zein mRNA: a region containing a duplicated AAUGAA poly(A) signal and a region that is present upstream from it. Strict spatial positioning of these two regions is required for efficient mRNA 3'-end processing. Insertion of a stuffer sequence as short as 17 or 18 bp either between the upstream region and the two AAUGAA motifs or between the two AAUGAA motifs drastically reduced the efficiency of 3'-end processing. Mutational analyses of the nucleotide preference at the fourth position of the AAUGAA motif revealed the preference order G > A >> C or U, suggesting that AAUAAA is neither a defective nor an optimal poly(A) signal for the 27-kDa zein mRNA. As for the 3' control region of the cauliflower mosaic virus (CaMV) transcription unit, the mRNA 3'-end processing mechanism mediated by the 27-kDa zein 3' control sequence is neither tissue nor species specific. The 3' upstream sequence of the 27-kDa zein gene can functionally replace that of the CaMV transcription unit. Conversely, the CaMV upstream sequence can mediate mRNA polyadenylation in the presence of a duplicated 27-kDa zein poly(A) signal. However, instead of the proximal poly(A) signal normally used in the 27-kDa zein mRNA, the distal signal is utilized. These results suggest that a general mechanism controls the 3'-end processing of plant mRNAs and that the cis-regulatory functions mediated by their upstream regions are interchangeable.

Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, the -300 element.

By utilizing a homologous transient-expression system, we have shown that a 58-bp sequence from the gamma-class 27-kDa zein promoter, spanning from -307 to -250 relative to the transcription start site, confers a high level of transcriptional activity on a truncated plant viral promoter. The transcriptional activity mediated by the 58-bp sequence is orientation independent, and it is further enhanced as a result of its multimerization. A similarly high level of transcriptional activity was also observed in protoplasts isolated from leaf tissue-derived maize suspension cells. In vitro binding and DNase I footprinting assays with nuclear protein prepared from cultured endosperm cells revealed the sequence-specific binding of a nuclear factor(s) to a 16-nucleotide sequence present in the 58-bp region. The nuclear factor binding sequence includes the -300 element, a cis-acting element highly conserved among different zein genes and many other cereal storage protein genes. A 23-bp oligonucleotide sequence containing the nuclear factor binding site is sufficient for binding the nuclear factor in vitro. It also confers a high level of transcriptional activity in vivo, but in an orientation-dependent manner. Four nucleotide substitutions in the -300 element drastically reduced binding and transcriptional activation by the nuclear factor. The same nuclear factor is abundant in the developing kernel endosperm and binds to the -300 element region of the 27-kDa or the alpha-class zein promoter. These results suggest that the highly conserved -300 element is involved in the common regulatory mechanisms mediating the coordinated expression of the zein genes.

Tissue-specific DNase l-sensitive sites of the maize P gene and their changes upon epimutation.

map has been developed of nuclease-hypersensitive sites of P-rr, the standard allele of the P-locus of Zea mays L. Using a traditional DNase I assay, eight such sites have been found that are specific for the expressing tissue and span a region of more than 25 kb of the P-locus, making it one of the largest plant genes yet described. The maps of the standard allele have also been compared with the recently described moderately stable P-pr allele, which arose from epimutation. Six of the eight sites exhibit the same tissue-specificity in P-pr plants, while two stay repressed as in non-expressing tissues of plants with the standard allele. Interestingly, the two repressed sites coincide with two hypermethylated restriction sites that have previously been correlated with the expression potential of the P-pr allele. On the other hand, four of the DNase I sites, coinciding with CpG islands that were not hypermethylated by the epimutation, also showed no differences in their sensitivity to DNase I between the standard allele and the P-pr allele. This suggests that the epimutation affects both site-specific methylation changes and a specific local chromatin structure of the P gene involved in its regulation.

Variegated phenotype and developmental methylation changes of a maize allele originating from epimutation.

Two instances of genetic transmission of spontaneous epimutation of the maize P-rr gene were identified. Transmission gave rise to two similar, moderately stable alleles, designated P-pr-1 and P-pr-2, that exhibited Mendelian behavior. Both isolates of P-pr conditioned a variable and variegated phenotype, unlike the uniform pigmentation conditioned by P-rr. Extensive genomic analysis failed to reveal insertions, deletions or restriction site polymorphisms between the new allele and its progenitor. However, methylation of the P gene was increased in P-pr relative to P-rr, and was greatly reduced (though not lost) in a revertant to uniform pigmentation. Variability in pigmentation conditioned by P-pr correlated with variability in transcript levels of the P gene, and both correlated inversely with variability in its methylation. Part of the variability in methylation could be accounted for by a developmental decrease in methylation in all tissues of plants carrying P-pr. We hypothesize that the variegated phenotype results from a general epigenetic pathway which causes a progressive decrease in methylation and increase in expression potential of the P gene as a function of cell divisions in each meristem of the plant. This renders all tissues chimeric for a functional gene; chimerism is visualized as variegation only in pericarp due to the tissue specificity of P gene expression. Therefore, this allele that originates from epimutation may exemplify an epigenetic mechanism for variegation in maize.

Characterization of a meiotic crossover in maize identified by a restriction fragment length polymorphism-based method.

Genetic map lengths do not correlate directly with genome size, suggesting that meiotic recombination is not uniform throughout the genome. Further, the abundance of repeated sequences in plant genomes requires that crossing over is restricted to particular genomic regions. We used a physical mapping approach to identify these regions without the bias introduced by phenotypic selection. This approach is based on the detection of nonparental polymorphisms formed by recombination between polymorphic alleles. In an F2 population of 48 maize plants, we identified a crossover at two of the seven restriction fragment length polymorphism loci tested. Characterization of one recombination event revealed that the crossover mapped within a 534-bp region of perfect homology between the parental alleles embedded in a 2773-bp unique sequence. No transcripts from this region could be detected. Sequences immediately surrounding the crossover site were not detectably methylated, except for an SstI site and at the flanking repetitive sequences were faithfully inherited by the recombinant allele. Our observations suggest that meiotic recombination in maize occurs between perfectly homologous sequences, within unmethylated, nonrepetitive regions of the genome.

Interchromosomal recombination in Zea mays.

A new allele of the 27-kD zein locus in maize has been generated by interchromosomal recombination between chromosomes of two different inbred lines. A continuous patch of at least 11,817 bp of inbred W64A, containing the previously characterized Ra allele of the 27-kD zein gene, has been inserted into the genome of A188 by a single crossover. While both junction sequences are conserved, sequences of the two homologs between these junctions differ considerably. W64A contains the 7313-bp-long retrotransposon, Zeon-1. A188 contains a second copy of the 27-kD zein gene and a 2-kb repetitive element. Therefore, recombination results in a 7.3-kb insertion and a 14-kb deletion compared to the original S+A188 allele. If nonpairing sequences are looped out, 206 single base changes, frequently clustered, are present. The structure of this allele may explain how a recently discovered example of somatic recombination occurred in an A188/W64A hybrid. This would indicate that despite these sequence differences, pairing between these alleles could occur early during plant development. Therefore, such a somatically derived chimeric chromosome can also be heritable and give rise to new alleles.

Maize glutamine synthetase cDNAs: isolation by direct genetic selection in Escherichia coli.

Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli delta glnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.

Region-specific cis- and trans-acting factors contribute to genetic variability in meiotic recombination in maize.

Understanding the genetic basis for variability in recombination rates is important for general genetic studies and plant-breeding efforts. Earlier studies had suggested increased recombination frequencies in particular F2 populations derived from the maize inbred A188. A detailed phenotypic and molecular analysis was undertaken to extend these observations and dissect the responsible factors. A heritable increase in recombination in the sh1-bz1 interval was observed in these populations. A factor causing an approximate twofold increase mapped to the A188 sh1-Bz1 region, behaved as a dominant, cis-acting factor, affected recombination equally in male and female sporogenesis and did not reduce the well-studied complete interference in the adjacent bz1-wx interval. This factor also did not increase recombination frequencies in the c1-sh1 and bz1-wx intervals, demonstrating independent control of recombination in adjacent intervals. Additional phenotypic analysis of recombination in the c1-sh1 and bz1-wx intervals and RFLP analysis of recombination along chromosomes 7 and 5 suggested that heritable factors controlling recombination in these intervals act largely independently and in trans. Our results show that recombination in these populations, and possibly maize in general, is controlled by both cis- and trans-acting factors that affect specific chromosomal regions.

Status of duckweed genomics and transcriptomics.

Duckweeds belong to the smallest flowering plants that undergo fast vegetative growth in an aquatic environment. They are commonly used in wastewater treatment and animal feed. Whereas duckweeds have been studied at the biochemical level, their reduced morphology and wide environmental adaption had not been subjected to molecular analysis until recently. Here, we review the progress that has been made in using a DNA barcode system and the sequences of chloroplast and mitochondrial genomes to identify duckweed species at the species or population level. We also review analysis of the nuclear genome sequence of Spirodela that provides new insights into fundamental biological questions. Indeed, reduced gene families and missing genes are consistent with its compact morphogenesis, aquatic floating and suppression of juvenile-to-adult transition. Furthermore, deep RNA sequencing of Spirodela at the onset of dormancy and Landoltia in exposure of nutrient deficiency illustrate the molecular network for environmental adaption and stress response, constituting major progress towards a post-genome sequencing phase, where further functional genomic details can be explored. Rapid advances in sequencing technologies could continue to promote a proliferation of genome sequences for additional ecotypes as well as for other duckweed species.

Chromatin organisation in duckweed interphase nuclei in relation to the nuclear DNA content.

The accessibility of DNA during fundamental processes, such as transcription, replication and DNA repair, is tightly modulated through a dynamic chromatin structure. Differences in large-scale chromatin structure at the microscopic level can be observed as euchromatic and heterochromatic domains in interphase nuclei. Here, key epigenetic marks, including histone H3 methylation and 5-methylcytosine (5-mC) as a DNA modification, were studied cytologically to describe the chromatin organisation of representative species of the five duckweed genera in the context of their nuclear DNA content, which ranged from 158 to 1881 Mbp. All studied duckweeds, including Spirodela polyrhiza with a genome size and repeat proportion similar to that of Arabidopsis thaliana, showed dispersed distribution of heterochromatin signatures (5mC, H3K9me2 and H3K27me1). This immunolabelling pattern resembles that of early developmental stages of Arabidopsis nuclei, with less pronounced heterochromatin chromocenters and heterochromatic marks weakly dispersed throughout the nucleus.

Cloning in M13 phage or how to use biology at its best.

A short historical account of the development of the M13mp phage vectors and of the pUC plasmids is presented. Moreover, a complete list of all the M13mp and pUC vectors is compiled.

New pUC-derived cloning vectors with different selectable markers and DNA replication origins.

Four new Escherichia coli cloning vectors are described, pUC6S, pUC21, pUK21 and pOK12. These vectors contain a polylinker or multiple cloning site (MCS) with the recognition sequences for 28 restriction enzymes. Plasmids pUC21, pUK21, and pOK12 contain the MCS in the N-terminal end of the lacZ alpha fragment allowing blue/white screening for inserts. To potentially increase the stability of some inserts that may encode toxic proteins, the strength of the lacZ promoter present on these vectors has been reduced. Plasmids pUC6S and pUC21 carry the bla gene encoding ampicillin resistance, while pUK21 and pOK12 contain the gene encoding kanamycin resistance. Plasmid pOK12 carries the replicon from P15A, resulting in a lower copy number pUC-type vector. Plasmid pUC6S carries the ori and bla gene present on all pUC vectors, but does not contain any lac sequences. Plasmids pUC21 and pUK21 contain the M13 intergenic region allowing for the production of plasmid single-stranded DNA. To improve the yield of ss plasmid DNA, two plasmid cis-acting factors that affect yield were also examined: the effect of plasmid-derived transcription across the M13 ori, and the effect of delecting the M13 minus-strand ori from the plasmid.

Modulation of gene expression by DNA-protein and protein-protein interactions in the promoter region of the zein multigene family.

A common cis-acting element in the promoter region of many genes expressed during endosperm development of cereal seeds, the prolamine-box or P-box, is only 20bp upstream of the alpha-class 22-kDa zein gene-specific cis element, the O2-box, which is recognized by the b-ZIP transcription factor, Opaque-2 (O2). The proximity of these two boxes has prompted a study of how two DNA-binding proteins of a different hierarchy might be involved in the activation and modulation of the 22-kDa zein-encoding genes. This was accomplished by utilizing a highly purified P-box-binding-factor-1 (PBF-1) and a bacterially expressed truncated form of the O2 protein. After adding the recombinant O2 to the purified fraction of PBF-1, binding studies were performed with a series of DNA probes combining the P- and O2-boxes from zein promoters. These studies have revealed an interesting inhibitory effect of PBF-1 over O2 function dependent on their ratio, consistent with its in-vivo properties and the developmental expression profiles of zein genes. We also could show that the P-box is specifically recognized by topoisomerase II and single-strand DNA-binding proteins, indicating a possible additional linkage between P-box and the scaffold-attachment-region (SAR).

The making of strand-specific M13 probes.

A novel approach has been developed for the preparation of highly radioactive, strand-specific M13 probes. A universal primer, complementary to the region 5' to the multiple cloning sites of M13mp7, was used to initiate the DNA synthesis of the complementary strand of the M13 sequence downstream from the inserted sequence. The synthesis of the (-) strand, which was labeled with a radioactively labeled precursor, did not proceed to completion so that the inserted sequence was kept single-stranded. Thus, a partially double-stranded probe that had the specificity of this inserted sequence was obtained. As an example for the application of single-stranded specific hybridization probes, an M13mp7 subclone of a zein cDNA clone of maize (A30) was labeled and used in a dot hybridization test to select from the hundreds of M13mp7 subclones of the zein genomic clone, Z4, the sequences complementary to the probe. The specificity of the probe was confirmed by dideoxy chain terminator sequencing experiments.

The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.

A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed. In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.

A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments.

The strategy of shotgun cloning with M13 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project. In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for M13 shotgun sequencing. Two new M13 vectors, M13mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (+) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M13mp8 and M13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.

Corrected nucleotide sequence of M13mp18 gene III.

There are seven differences between the actual nucleotide (nt) sequence of bacteriophage M13mp18 gene III and the previously reported nt sequence (which had been compiled based on the nt sequence of wild-type bacteriophage M13 gene III).

Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.

The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M13mp10 and M13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.

A versatile primer for DNA sequencing in the M13mp2 cloning system.

A primer for DNA sequencing by the chain-termination method in the M13mp2 cloning system was constructed and amplified. The primer was isolated as an EcoRI/AluI restriction fragment. After conversion of the AluI end into an EcoRI end the fragment was cloned in pBR325 from which it can be recovered by cleavage with EcoRI. The primer hybridizes to the single-stranded DNA of the mature M13mp2 phage next to the site of insertion thereby directing DNA synthesis along the inserted DNA.