Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis

Intron-bearing replacement histone H3 genes in Arabidopsis and other plants are highly and constitutively expressed. We demonstrate that the introns located within the 5-untranslated regions (5-UTR) of the two Arabidopsis replacement H3 genes will abolish the cell cycle dependence of an endogenous histone H4 promoter. We demonstrate that these introns, functionally combined with their endogenous promoters, could produce the high and constitutive expression of the replacement H3 genes observed in planta. They strongly increase gene expression whatever the promoter, from the strong 35S CaMV promoter to complete and resected promoters of cell cycle-dependent and replacement histone genes. Quantitative analysis of the extent of reporter gene enhancement in different parts of developing transgenic plantlets, ranging from 2-fold to 70-fold, supports the notion that trans-acting factors are responsible for this effect. Such factors appear most abundant in roots.     

The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization, evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5 border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3 acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

Intron-containing tRNA genes ofSulfolobus solfataricus

Summary Nucleotide sequences of four tRNA genes from the archaebacteriumSulfolobus solfataricus have been determined. Based upon DNA sequence analysis, three of the four genes contain presumptive intervening sequences (introns) in their anticodon loops. The three introns can form similar, but not identical, secondary structures. The cleavage site at the 3 end of all three introns occurs in a three-base bulge loop. All four genes lack an encoded 3 CCA terminus and are flanked by A+T-rich DNA sequences. Two of the genes are located on antiparallel DNA strands, with their 3 termini separated by 414 bp of sequence. Including two previously published sequences, a total of five introns have now been detected among sixS. solfataricus tRNA genes. Occurrence of introns at corresponding locations in both archaebacterial and eukaryotic tRNA genes suggests that the intron/exon form of gene structure predates the evolutionary divergence of the archaebacteria and the eukaryotes.     

Identification of a multigene family for small heat shock proteins in soybean and physical characterization of one individual gene coding region

When soybean seedlings are tranferred from 28 to 40 ° C, a heat shock (hs) response is elicited. This is characterized by the synthesis of a new set of proteins (hs-proteins) and by cessation of normal protein synthesis (8). At the level of poly(A)mRNA, a new class of highly abundant RNAs appears which encodes a group of hs-proteins in the low molecular weight range of 15–18 kD (11). The classification of these proteins/genes into several sub-classes is based on a complex sequence relationship for class I protein/genes.This was confirmed by both the complexity and the similarity of southern blot hybridization patterns of genomic DNA digests with class I cDNA-probes. Genomic DNA clones (obtained from -libraries by screening with cDNA-probes) for the class I gene 1968 showed cross hybridization with all other class I cDNA-probes. Higher specificity of gene/protein correlation was obtained by variation of hybridization criteria. The specificity of cDNA clone 1968 for the genomic DNA clone hs68-7 was demonstrated by thermal stability of hybridization at 55 ° C and 65 ° C in 50% formamide compared to other cross-reacting probes. The correlation of clone 1968 with a specific hs-protein was obtained by temperature dependent release of hybrid selected hs-mRNAs at 50, 60, 70 and 85 ° C followed byin vitro translation and two-dimensional gel analysis. The coding regions of hs-genes on genomic DNA clones were mapped by R-loop formation. The position of R-loops was mapped relative to certain restriction sites on subclones of hs68-7 DNA. The polarity of hs-genes was determined by attaching X174RF-DNA labels to the 3 poly(A)-tails of the mRNAs of R-loops.     

The pea lectin gene family contains only one functional gene

Molecular hybridization experiments have shown that the pea genome contains four regions which hybridize with pea lectin cDNA (Kaminski, Buffard, and Strosberg, 1986. Plant Science 46, 111–116). The complete organization of the pea lectin gene family was investigated. Four partial EcoRI genomic libraries were screened with a lectin cDNA (pPS 15–50) covering the entire coding region. Four positive recombinant phages, I 101, I 52, III 51 and IV 22, were isolated and the DNA sequences of the subclones, designated respectively PSL1, PSL2, PSL3 and PSL4, were determined. PSL2, PSL3 and PSL4 are incomplete genes; the presence of several stop codons in the correct reading frames indicate that these genes cannot code for a functional lectin protein. The sequences of PSL1 and pPS 15–50 have identical coding regions. The pea lectin gene has no intervening sequences and is flanked at its 5 region by a sequence containing an exceptionally high A+T content (73%). Eucaryotic consensus sequences such as a TATA box and a polyadenylation signal are also found in the flanking regions of the PSL1 clone.     

Structural analysis of a maize gene coding for glutathione-S-transferase involved in herbicide detoxification

Summary We have used the cDNA clone encoding maize glutathione-S-transferase (GST I) to isolate a genomic DNA clone containing the complete GST I gene. Nucleotide sequence analysis of the cDNA and genomic clones has yielded a complete amino acid sequence for maize GST I and provided the exon-intron map of its gene. The mRNA homologous sequences in the maize GST I gene consist of a 107 bp 5 untranslated region, a 642 bp coding region and 340 bp of the 3 untranslated region. They are divided into three exons by two introns which interrupt the coding region. The 5 untranslated spacer contains an unusual sequence of pentamer AGAGG repeated seven times. The inbred maize line (Missouri 17) contains a single gene for GST I, whereas the hybrid line (3780A) contains two genes. Nucleotide sequence analysis of the primer extended cDNA products reveals that the 5 untranslated regions of the two genes in the hybrid 3780A are identical except for a 6 bp internal deletion (or insertion). The amino acid sequence of maize GST I shares no apparent sequence homology with the published sequences of animal GST's and represents the first published sequence of a plant GST. re]19850813 ac]19851126     

Organization and characterization of Cucurbita phloem lectin genes

The phloem of pumpkin and squash contains a dimeric chitin-binding lectin called PP2 (phloem protein 2). We have isolated three genomic clones from pumpkin (Cucurbita maxima Duch.) that encoded PP2. One clone, gPC13-1, contained two PP2 genes that were 99.8% identical over a region of 3055 nucleotides. This conserved region included 1922 bp of 5 non-coding sequence, 844 bp of protein coding sequence (including two introns), and 289 bp of 3 non-coding sequence. To examine the conservation of the phloem lectin within the genus Cucurbita, we analyzed nine different species for PP2, its mRNA, and the genes that encode PP2. DNA blot analysis indicated that each species contained genes that encoded PP2, however, there was considerable restriction fragment length polymorphism (RFLP) among the species. PP2 gene copy number reconstructions indicated that PP2 is encoded by a small gene family (two to eight genes). Although a high level of PP2 DNA polymorphism existed among species, a single mRNA (ca. 1 kb) was detected in each species. PP2, affinity-purified from the vascular exudate of each species, reacted with PP2-specific antibodies; five species contained a single PP2 polypeptide while four species contained two PP2 polypeptides.     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Nucleotide sequence of the histone gene cluster in the coral acropora formosa (cnidaria; scleractinia): Features of histone gene structure and organization are common to diploblastic and triploblastic metazoans

We report the nucleotide sequence of the core histone gene cluster from the Cnidarian Acropora formosa. This is the first histone gene cluster to be sequenced from a diploblastic organism and the predicted amino acid sequences most resemble those of sea urchin equivalents. Each of the Cnidarian histone genes has two conserved regions 3 of the coding sequences and these closely resemble those of the metazoan a-class histone genes. In A. formosa the core histone genes are arranged as opposed (H3/H4 and H2A/H2B) pairs, a pattern common to the nondeuterostome metazoa, and tandem repetition is the predominant pattern of organization in the Cnidarian. With the recent identification of several classes of homeobox genes in Cnidarians these features clearly align the Cnidaria with triploblastic metazoans, supporting a monophyletic origin of the metazoa.     

Histone H4 and H2B genes in rainbow trout (Salmo gairdnerii)

Summary The complete nucleotide sequence of the 3.0-kb BamH I-Sst I restriction fragment contained within the rainbow trout genomic clone TH2 has been determined. This region contains the rainbow trout H4 and H2B histone genes and 5 and 3 flanking and spacer sequences, and represents the 5 half of the histone-gene cluster; the remaining half has been characterized previously. The genes are uninterrupted, and are transcribed from the same strand. The protein sequence of H4, as determined from the nucleic acid sequence, is the same as that derived for other vertebrate H4 proteins, although comparison of nucleotide sequences shows a great deal of sequence divergence, especially in the third base position. The amino acid sequence of H2B, though largely homologous to those of other vertebrate H2B proteins, displays some characteristic differences in primary structure. Consensus sequences noted in many other eukaryotic genes, as well as histone-specific consensus sequences, have been identified. An unusual feature of the spacer region between the H4 and H2B genes is the presence of a duplicated sequence 87 bp in length. The 5 and 3 ends of each repeat are complementary, and each repeat contains smaller repeated sequences internally, as well as a possible cruciform structure.