Isolation and sequence analysis of a barley alpha-amylase cDNA clone

We have isolated a cDNA clone derived from poly(A+) RNA from barley aleurone cells stimulated with gibberellic acid. This cDNA clone contains one open reading frame coding for 438 amino acids. The cloned DNA hybridizes to a poly(A+) RNA species 1550 bases in size, the same size as the most abundant poly(A+) RNA molecules in stimulated cells. RNA complementary to this clone can be translated to make immunoprecipitable alpha-amylase in the wheat germ system and increases about 5-fold in quantity after gibberellic acid stimulation of aleurone cells. In contrast, hybridization experiments using a total cDNA probe demonstrate that the most abundant mRNA population, identical in size with our cloned sequence and presumably that for alpha-amylase, increases at least 17-fold after gibberellic acid stimulation. We therefore infer that there must be at least two populations of alpha-amylase mRNA molecules derived from separate structural genes differently influenced by gibberellic acid in aleurone cells.     

Barley alpha-amylase genes. Quantitative comparison of steady-state mRNA levels from individual members of the two different families expressed in aleurone cells

We have cloned and sequenced two barley alpha-amylase genes belonging to the high pI isozyme family, one of which, Amy6-4, corresponds to a cDNA previously characterized by our laboratory. A 750-base pair probe from Amy6-4, representing primarily the promoter/upstream sequences cross-hybridizes on genomic Southern blots under stringent conditions to five other genes or pseudogenes; this demonstrates that the promoter/upstream region in these different members of the gene family is highly conserved. In contrast, this probe hybridizes very poorly to the genomic fragment containing the other cloned high pI gene, Amy46, a finding consistent with substantial divergence of sequence about 200 base pairs upstream from the TATA box of each. We compared steady-state mRNA levels from these individual genes to levels for mRNAs from two low pI alpha-amylase genes and from the single copy gene for aleurain, a thiol protease, using quantitative S1 nuclease protection assays. We found, in RNA from aleurone cells treated with gibberellic acid for 19-24 h, that the two low pI alpha-amylase mRNAs are each about five times more abundant than Amy6-4 or aleurain, which are, in turn, about 10 times more abundant than Amy46. These results indicate that as many as seven closely related high pI genes are needed to provide mRNA levels approaching those from the two low pI genes. We speculate that the substantially lower level of expression of Amy46 may be related to its divergent sequence upstream from the promoter.     

A uniquely conserved regulatory signal is found around the translation initiation site in three different collagen genes

We have determined the nucleotide sequence of the 5' untranslated region and the sequence encoding the signal peptide for mRNAs of the chick alpha 1 type I and alpha 1 type III collagen. These sequences were obtained by synthesizing the corresponding cDNAs using as primers either a synthetic oligonucleotide to prime alpha 1 type I cDNA or a DNA fragment isolated from a genomic clone coding for alpha 1 type III collagen to prime the cognate cDNA. Both primers were selected so that the resulting cDNAs would be short and would contain sequence information for the 5' untranslated region and the signal peptide of the proteins. The nucleotide sequences of these cDNAs were compared with the corresponding sequence of alpha 2 type I collagen. In each mRNA the 5' untranslated segment is approximately 130 nucleotides and contains two or more AUG triplets preceding the AUG which serves as a translation initiation codon. A sequence of about 50 nucleotides surrounding the translation initiation codon is remarkably conserved in all three mRNAs, whereas the sequences preceding and following this segment diverge markedly. This homologous sequence contains an almost identical inverted repeat sequence which could form a stable stem-loop structure. The initiation codon and the AUG which precedes it are found at the same place within this symmetrical sequence and the distance between them is invariant. The rest of the conserved sequence shows a less perfect symmetry. This conserved sequence has not been found in other genes. Our data suggest that these three and perhaps other collagen genes contain an identical regulatory signal that may play a role in determining the level of expression of these genes by modulating translational efficiency.     

Molecular characterization of the interferon-induced 15-kDa protein. Molecular cloning and nucleotide and amino acid sequence

We have isolated a cDNA clone for an interferon-induced 15-kDa protein. The cDNA clone was prepared from mRNA isolated from interferon-beta-treated human Daudi cells. The clone of 635 base pairs contains an open reading frame coding for a protein of 145 amino acids, and suggests for the mRNA a 75-base pair 5' untranslated and a 125-base pair 3' untranslated region. Approximately 85% of the amino acid sequence of the 15-kDa protein has been independently obtained from 2 nmol of material using microsequencing technology on the N terminus of the intact protein and on tryptic and chymotryptic peptides. The amino acid sequence of the isolated protein is identical to the amino acid sequence deduced from the cDNA. Northern blot analysis confirmed that the mRNA for the 15-kDa protein is undetectable in untreated cells, but is greatly induced following interferon treatment.     

Nucleotide sequence analysis of alpha-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells.

We have determined the nucleotide sequences of Amy32b, a type A alpha-amylase gene, and of the gene for aleurain, a thiol protease closely related to mammalian cathepsin H. Both are expressed in barley aleurone cells under control of the plant hormones gibberellic acid and abscisic acid, but only aleurain is expressed at high levels in other barley tissues. Sequence analysis indicates that the 5' end of the aleurain gene, comprising 3 exons and 2 introns, may have become associated with the remainder of the gene, encoding the protease domain of the protein, by some sort of recombination event. This 5' domain of the gene is very G + C-rich and is flanked by inverted repetitive sequences. We found two different groups of homologous sequence elements. The first group consists of four blocks of sequences conserved in the same spatial arrangement in both genes; these are arranged at similar intervals upstream from the Amy32b TATA box and from a TATA box present in intron 3 of aleurain, outside of the 5' domain and upstream from the protease domain. A part of two of these conserved sequences is similar to the core sequence of certain enhancer elements characterized from mammalian cells. The second group of homologous elements is present in the upstream region of both genes. We speculate that these conserved sets of sequences may have some role in either the tissue specificity of expression of the genes or in some part of the hormonal regulation imposed on them.     

The keratin BIIIB gene family: isolation of cDNA clones and structure of a gene and a related pseudogene

The nucleotide sequence of cDNA clones encoding the three major BIIIB high-sulfur wool keratin proteins (BIIIB2, 3, and 4) and the structure of a BIIIB4 gene and a BIIIB3 pseudogene are reported. Although Southern blot analysis indicates that the BIIIB genes comprise a multigene family in the sheep genome, they are poorly represented in genomic DNA libraries. The family sequence homology of the coding region extends into the 5' and 3' untranslated regions and the near 5' flanking region of the BIIIB3 and 4 genes. These homologies suggest that the BIIIB3 and 4 genes represent the latest gene duplication event in the evolution of the BIIIB multigene family. Like the genes coding for other wool keratin matrix protein components, the BIIIB genes have the conserved 18-bp sequence immediately 5' to the initiation codon and also appear to lack introns.