Maize Genomic DNA Research Articles

Two genes encode the adenine nucleotide translocator of maize mitochondria

Southern blot hybridisation of maize (Zea mays) genomic DNA with a specific cDNA probe and sequence analysis of several cDNAs have revealed that the mitochondrial adenine nucleotide translocator (ADP/ATP carrier) is encoded by two genes, both of which are expressed in dark-grown coleoptile tissue. The structural genes (designated ANT-G1 and ANT-G2), encoding the two translocator polypeptides, have been isolated from a maize genomic library and characterised by DNA sequence analysis, mung bean nuclease mapping and primer extension. The two genes each contain two introns and encode very similar proteins. Transcripts from both genes have long (approximately 200 nucleotides) 5' untranslated leaders which are unusual in that they contain several ATG codons upstream of the initiation codon. Transcript analysis of RNA extracted from serial sections of developing maize leaves reveals that the translocator genes are most actively transcribed in the basal meristem, while transcripts were undetectable in green leaves.

Maize pyruvate decarboxylase mRNA is induced anaerobically

A cDNA was identified using an oligonucleotide designed by comparing the sequences of bacterial and yeast pyruvate decarboxylase. The sequence of the cDNA identified by the oligonucleotide contained an open reading frame that encoded a protein of 65 kDa that was similar in sequence to bacterial and yeast pyruvate decarboxylase. This protein was selectively precipitated by an antiserum specific for maize PDC. Northern-blot analysis shows that PDC mRNA is anaerobically induced. Southern-blot analysis of maize genomic DNA indicated that the maize PDC gene has a single or low copy number.

Characterization of catalase transcripts and their differential expression in maize

In maize, the three unlinked catalase (EC 1.11.1.6) structural genes ( Cat1, Cat2 and Cat3) are differentially expressed temporally, spatially and in response to environmental signals in the developing seedling. In order to understand more fully the molecular mechanisms involved in catalase gene expression, full-length cDNA clones representing the maize Cat1, Cat2 and Cat3 transcripts were isolated and characterized. DNA sequence analysis confirmed that each cDNA encodes a unique catalase protein. Gene-specific probes for the three maize catalase cDNAs were isolated and used to probe blots of poly(A) + RNA isolated from various maize tissues. Cat1 mRNA was found in scutella, milky endosperm of immature kernels, leaves and epicotyls. The Cat2 mRNA was present primarily in post-germinative scutella, with lower levels in leaves and epicotyls. Cat3 mRNA was detected primarily in epicotyls and, to a lesser extent, in leaves and scutella. The gene-specific probes hybridized with maize genomic DNA blots in simple, but unique patterns, indicating that there is one, or a very few copies of each catalase gene. The coding region of the Cat3 cDNA comprised 66% G + C, which led to a strong codon usage bias in this gene. This codon bias was also seen with the Cat2 transcripts, but not with those for Cat1. A high degree of similarity was found between the maize catalase nucleic acid and deduced amino-acid sequences and those of sweet potato and rat liver catalase.

Expression of lipid body protein gene during maize seed development. Spatial, temporal, and hormonal regulation.

The storage lipids of seeds are packaged into lipid bodies, simple organelles containing a triacylglycerol core surrounded by a layer of phospholipids and proteins. A cDNA encoding the major protein from lipid bodies of maize (L3) was used as a hybridization probe to analyze the regulation of lipid body biosynthesis during seed development and germination. Immunofluorescent microscopy demonstrates that L3 protein accumulation is tissue-specific, confined to the embryo (scutellum and embryonic axis) and the aleurone layer of developing seed. Northern analyses show that L3 mRNA also accumulates to high levels in the embryo and is not found in any nonseed tissue. The steady-state level of L3 mRNA is developmentally regulated, increasing during early seed development and peaking at about the midpoint of seed development. It then decreases slowly, to 20% of the peak level, in the embryo of the mature seed and declines rapidly to undetectable levels as the seed germinates. The high level of L3 gene expression during seed development is not due to amplification of the L3 gene, since Southern analyses of maize genomic DNA indicate that the L3 gene has the same relatively low copy number throughout development (1-4 copies/haploid) genome). Rather, the increase in the steady-state level of L3 mRNA during seed development is transcriptionally regulated. The developmental changes in L3 mRNA levels are paralleled by changes in the transcription of the L3 gene, as measured by run-off transcription in isolated nuclei. The role of the plant growth regulator abscisic acid in regulating L3 gene expression was investigated. When the mature seed is allowed to imbibe in the presence of exogenous abscisic acid, germination is retarded, and the period of L3 gene expression is extended. The extended period of L3 gene expression in the presence of exogenous abscisic acid is due, at least in part, to a dramatic and specific increase in L3 gene transcription.

Genomic organization of two families of highly repeated nuclear DNA sequences of maize selected for autonomous replicating activity in yeast

Maize nuclear DNA sequences capable of promoting the autonomous replication of plasmids in yeast were isolated by ligating Eco RI-digested fragments into yeast vectors unable to replicate autonomously. Three such autonomously replicating sequences (ARS), representing two families of highly repeated sequences within the maize genome, were isolated and characterized. Each repetitive family shows hybridization patterns on a Southern blot characteristic of a dispersed sequence. Unlike most repetitive sequences in maize, both ARS families have a constant copy number and characteristic genomic hybridization pattern in the inbred lines examined. Larger genome clones with sequence homology to the ARS-containing elements were selected from a lambda library of maize genomic DNA. There was typically only one copy of an ARS-homologous sequence on each 12-15 kb genomic fragment.

Maize phosphoenolpyruvate carboxylase. Cloning and characterization of mRNAs encoding isozymic forms.

The isozymic forms of maize phosphoenolpyruvate carboxylase (P-enolpyruvate carboxylase) involved in photosynthetic CO2 fixation were shown by protein gel blot analysis to consist of 100-kDa subunits. The nonautotrophic isoform found in roots is comprised of 96-kDa subunits and is about 50-100-fold less prevalent. Further analysis of P-enolpyruvate carboxylase isoforms made use of cloned cDNA probes. Two cDNA clones were isolated from a library constructed from maize leaf poly(A) RNA. The largest clone was complementary to about 25% of P-enolpyruvate carboxylase mRNA, which is 3.4 kilobases in length. The quantity of P-enolpyruvate carboxylase mRNA in green, mature leaf tissue was estimated to be 0.20% of poly(A) RNA, whereas P-enolpyruvate carboxylase mRNA in roots was about 100-fold less prevalent. We used thermal denaturation of a P-enolpyruvate carboxylase cDNA probe hybridized to RNA gel blots to estimate the degree of sequence difference between mRNAs encoding different P-enolpyruvate carboxylase isoforms. There appear to be at least two prevalent P-enolpyruvate carboxylase mRNAs in green leaves which are significantly different in sequence, as are P-enolpyruvate carboxylase mRNAs in roots and shoots. The hybridization pattern of maize genomic DNA Southern blots indicates that P-enolpyruvate carboxylase is encoded by a small gene family.