Irwin Rubenstein

Plant Gene Structure

Techniques in molecular cloning and DNA sequencing have provided the tools to study the structure of genes at the nucleotide level. Most of these studies have been conducted on mammalian genes. From the comparison of individual genes much knowledge has been gained about organization and potential signal sequences. Although relatively little sequence data is available for plant genes, their number has grown to a degree where similar comparisons may be initiated. Using our studies of the zein storage protein and the data of other laboratories we may draw the following conclusions: 1) Like other plant genes, zein genes are organized in multigene families. Hybridization techniques and sequence data further subdivide the zein multigene family into subfamilies. 2) The sequence data obtained also allows us to determine the protein sequence and the sequence variation among the zein proteins which has occurred during evolution. 3) Comparing the zein genes and other known plant genes, we have identified potential signal sequences which can be distinguished from those of animal genes.

Complex organization of zein genes in maize

We have examined the fragments of maize nuclear DNA that are homologous to three cloned cDNAs prepared from zein mRNA. Southern blots of high molecular weight (greater than 40 kb) maize nuclear DNA cleaved with BamHI, HindIII or EcoRI were hybridized to three zein cDNA plasmid probes. Multiple restriction fragments in a wide range of size classes were observed to hybridize with all three probes. Our results indicate the occurrence of families of genes in the maize genome that are related by their sequences to a given zein mRNA sequence.

Primary structure of a genomic zein sequence of maize.

The nucleotide sequence of a genomic clone (termed Z4) of the zein multigene family was compared to the nucleotide sequence of related cDNA clones of zein mRNAs. A tandem duplication of a 96‐bp sequence is found in the genomic clone that is not present in the related cDNA clones. When the duplication is disregarded, the nucleotide sequence homology between Z4 and its related cDNAs was approximately 97%. The nucleotide sequence is also compared to other isolated cDNAs. No introns in the coding region of the zein gene are detected. The first nucleotide of a putative TATA box, TATAAATA, was located 88 nucleotides upstream of the first nucleotide of the first ATG codon which initiated the open reading frame. The first nucleotide of a putative CCAAT box, CAAAAT, appeared 45 nucleotides upstream of the first nucleotide of the zein cDNA clones in the 3′ non‐coding region also appeared in the genomic sequence at the same locations. The amino acid composition of the polypeptide specified by the Z4 nucleotide sequence is similar to the known composition of zein proteins.

Localization of the 5S rRNA genes and evidence for diversity in the 5S rDNA region of maize.

Abstract The 5S rRNA genes of maize are located in the long arm of chromosome 2 (88% of the distance from the centromere to the end) and organized in a 320-bp repeat. Genomic blots of maize DNA digested with the restriction enzymes Bam HI and Msp I reveal ladders of bands in multiples of 320 bp. Analysis of 5S rDNA from genomic clones reveals that the ladders are due to both modification and divergence of the 5s rDNA nucleotide sequence.

Sequence analysis and comparison of cDNAs of the zein multigene family .

The nucleotide sequence of two zein cDNAs in hybrid plasmids A20 and B49 have been determined. The insert in A20 is 921 bp long including a 5′ non‐coding region of 60 nucleotides, preceded by what is believed to be an artifactual sequence of 41 nucleotides, and a 3′ non‐coding region of 87 nucleotides. The B49 insert is 467 bp long and includes approximately one‐half the protein coding sequence as well as a 3′ non‐coding region of 97 nucleotides. These sequences have been compared with the previously published sequence of another zein clone, A30 . A20 and A30, both encoding 19 000 mol. wt. zeins, have approximately 85% homology at the nucleotide level. The B49 sequence, corresponding to a 22 000 mol. wt. zein, has approximately 65% homology to either A20 or A30 . All three zeins share common features including nearly identical amino acid compositions. In addition, the tandem repeats of 20 amino acids first seen in A30 are also present in A20 and B49 .

Two-dimensional gel analysis of the zein proteins in maize.

Abstract The level of complexity of the polypeptide components in the zein (ethanol soluble) fraction from maize kernels can be shown clearly by an analysis that combines the techniques of isoelectric focusing and SDS gel electrophoresis. This high resolution system gives unique and repeatable gel patterns of the zein fraction from 6 maize lines. The gel patterns of zein from normal lines are very complex with over 10 components resolved. The patterns from lines containing the o2 mutation are less complex, with a reduction in the number of components that can be resolved, especially within the 22 500 mol. wt class.

Transcriptional characterization of an α-zein gene cluster in maize

A cluster of five α-zein subfamily 4 (α-zein SF4) genes are present in a 56 kb region of the maize W22 genome. Two types of α-zein SF4 genes are in the cluster. One of the genes, termed a type 1 (T1) α-zein SF4 gene, contains no early in-frame stop codons. Four of the genes, termed type 2 (T2) α-zein SF4 genes, contain one or two early in-frame stop codons. The base sequence of the T1 α-zein SF4 gene is similar (>90%) to the sequences of any of the four T2 α-zein SF4 genes. However, their sequences differ markedly at distances greater than -875 bp upstream from the translation initiation codon of the α-zein coding region. This region of dissimilarity is well inside the functional 5′-flanking region for the genes since a 1.8 kb transcript is initiated in this region and the sequences of the T2 α-zein SF4 genes are similar in this region. Two sizes of mRNA transcripts, 1.8 kb and 0.9 kb, were detected in a gene specific manner for 4 of the 5 genes in this α-zein SF4 gene cluster. One of the T2 α-zein SF4 genes had only the 0.9 kb transcript. The RNA level for the 0.9 kb transcript of the T1 α-zein SF4 gene was 5- to 10-fold higher than the transcript levels of any of the T2 α-zein SF4 genes. In each case, the amount of the 0.9 kb transcript detected was at least 5-fold higher than the amount of the 1.8 kb transcript. A cDNA clone with a sequence identical to a T2 α-zein SF4 gene was isolated, providing the first direct evidence for the transcription of T2 α-zein genes containing early in-frame stop codon(s) in maize endosperm.

Molecular characterization of two types of 22 kilodalton α-zein genes in a gene cluster in maize

SummaryFive genes of the α-zein subfamily four (SF4) are located in a 56 kb genomic region of the maize inbred line W22. Their nucleotide and deduced amino acid sequences have been determined. The sequences define two types of α-zein SF4 genes — type 1 (T1) and type 2 (T2). The single T1 α-zein SF4 gene codes for an α-zein protein with a Mr of about 22 000. This is the first α-zein SF4 gene sequenced that contains no early in-frame stop codons in its coding sequence. The four T2 α-zein SF4 genes in this cluster contain one or two early in-frame stop codons. In addition, our T1 and T2 genes differ markedly in the base sequences of their distal 5′ non-translated flanking regions. The nucleotide and the deduced amino acid sequences of these two types of α-zein SF4 genes are similar ( > 90 %) to one another and to all known α-zein SF4 genes and cDNAs. Of the known W22 α-zein SF4 genes, only one in six does not contain an early in-frame stop codon. If the number of α-zein SF4 genes is 15–20, then we estimate that only about 4 of the W22 α-zein SF4 genes are without in-frame early stop codons.

Cloned genomic segments of Zea mays homologous to zein mRNAs

Abstract A maize genome library was constructed using maize W22 DNA from leaf tissue nuclei and bacteriophage λCh4 as the vector. cDNA clones of zein mRNA were used to identify homologous genomic sequences in the Ch4 maize library. Each of the genomic clones identified has homology to a family of mRNAs in the zein mRNA population. This paper reports on the construction of the library, the isolation of the genomic clones and their partial characterization.

Genomic organization of an α-zein gene cluster in maize

SummaryThe genes encoding the α-zein proteins of maize constitute a large multigene family of some 75 genes. This multigene family can be divided into four subfamilies based on the nucleotide sequences of their genes and the deduced amino acid sequences of their proteins. We describe for the first time evidence of a clustering of five α-zein subfamily 4 (SF4) genes that are members of one of the major α-zein subfamilies in a 56 kb region of the genome of the maize inbred line W22. None of the other three known α-zein gene subfamilies (SF1, SF2, or SF3) are present in this cluster. The genomic region was reconstructed using restriction endonuclease maps to identify and align three overlapping cosmid clones isolated from a genomic library. The α-zein genes are not evenly spaced; the minimum distance between genes is 3.5 kb; the maximum is 13 kb. All the α-zein genes in the cluster have the same transcriptional orientation. The location and sequences of some of the repetitive DNA elements in this gene cluster were determined. We estimate that there are a minimum of eight repetitive DNA elements in this region. The sequences of the repetitive elements (not functionally defined) are located between or among the α-zein genes. The regions containing two of these repetitive elements (Rep1 and Rep4) have been sequenced; they are about 15 kb apart in the genome. These repetitive elements have similar sequences for about 300 by out of the 400 by compared. The regions of sequence similarity, however, are in reverse orientation to one another. Both repetitive elements contain replication origin-like sequences. In addition, Rep4 contains two repeats of a five-base sequence that appear to define its presumptive ends. The presence of the short direct repeats flanking the Rep4 element suggests that the Rep4 element might have originated from a transposition event.