Applications of an optimized monocot expression vector in studying transient gene expression and stable transformation of barley

Protoplasts of a barley ( Hordeum vulgare L. cv. Golden Promise) suspension cell line were used for PEG-mediated gene transfer. Transient gene expression in barley protoplasts was studied using a chimeric CaMV 35S cat construct, which was only poorly expressed in barley cells. However, insertion of exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene in the 5'-untranslated leader of the construct strongly stimulated gene expression. By using the optimized chimeric cat construction the amount of CAT protein that was reached 19 hours after DNA uptake was 0.5% of total protein, which was calculated from western blot data.
As an alternative marker gene for expression studies, we also tested the firefly luciferase gene in barley protoplasts. Low level expression of chimeric CaMV 35S luciferase genes could be highly stimulated when Sh1 exon1 and intron1 were inserted in the 5'-untranslated leader of the constructs. Enhanced luciferase gene expression by Shrunken-1 intronic sequences enabled us to monitor gene integration events early after DNA uptake using a promoterless luciferase marker gene, which could only be expressed after integration behind an endogenous promoter.     

Intron-mediated enhancement of heterologous gene expression in maize

Chimeric genes containing the coding sequence for bacterial chloramphenicol acetyl transferase (CAT) have been introduced by electroporation into maize protoplasts (Black Mexican Sweet) and transient expression monitored by enzyme assays. Levels of CAT expression were enhanced 12-fold and 20-fold respectively by the inclusion of maize alcohol dehydrogenase-1 introns 2 and 6 in the chimeric construct. This enhancement was seen when the intron was placed within the 5 translated region but not when it was located upstream of the promoter or within the 3 untranslated region. Deletion of exon sequences adjacent to intron 2 abolished its ability to mediate enhancement of CAT gene expression. Northern analysis of protoplasts electroporated with intron constructs revealed elevated levels of CAT mRNA. However, this elevation was insufficient to account for the increased enzyme activity. One explanation of these results is that splicing affects both the quantity of mRNA.     

Nucleotide sequence of the chicken cardiac alpha actin gene: absence of strong homologies in the promoter and 3'-untranslated regions with the skeletal alpha actin sequence

The entire nucleotide sequence of the chicken cardiac alpha-actin (CC alpha A) gene has been determined. This is the first complete sequence of a cardiac actin gene that includes the promoter region, cap site, all the introns, and the polyadenylation site. The gene contains six introns, five of which interrupt the coding region at amino acids (aa) 41, 150, 204, 267, and 327. The first intron is in the 5'-noncoding region and is 438 bp in length. The CC alpha A gene encodes an mRNA of approx. 1400 bp with 5'- and 3'-untranslated region of 59 and 184 nucleotides (nt), respectively. Like the chicken skeletal alpha-actin gene, the CC alpha A gene has the codon for the aa cysteine between the initiator ATG and the codon for the N-terminal aspartic acid residue of the mature protein. There are no strong homologies (less than 13 consecutive nt) in the promoter or 3'-untranslated regions between the CC alpha A and chicken skeletal alpha-actin genes even though both are expressed in skeletal muscle during development. However, the 3'-untranslated region of the CC alpha A gene demonstrates significant sequence homology (76% over a 200-nt region) with the same region in the partial sequence of the human cardiac gene. The conservation of these sequence homologies between identical isoforms rather than the different alpha actin genes suggests these conserved regions may have a role in regulation rather than tissue-specific expression, as previously proposed.     

Tissue-preferential expression of a rice alpha-tubulin gene, OsTubA1, mediated by the first intron

The genomic clone encoding an alpha-tubulin, OsTubA1, has been isolated from rice (Oryza sativa L.). The gene consists of four exons and three introns. RNA-blot analysis showed that the gene is strongly expressed in actively dividing tissues, including root tips, young leaves, and young flowers. Analysis of chimeric fusions between OsTubA1 and beta-glucuronidase (GUS) revealed that the intron 1 was required for high-level GUS expression in actively dividing tissues, corresponding with normal expression pattern of OsTubA1. Fusion constructs lacking the intron 1 showed more GUS staining in mature tissues rather than young tissues. When the intron 1 was placed at the distal region from 5'-upstream region or at the 3'-untranslated region, no enhancement of GUS expression was observed. Sequential deletions of the OsTubA1 intron 1 brought about a gradual reduction of GUS activity in calli. These results suggest that tissue-preferential expression of the OsTubA1 gene is mediated by the intron 1 and that it may be involved in a mechanism for an efficient RNA splicing that is position dependent.     

Thymidylate synthase gene expression is stimulated by some (but not all) introns.

We previously described the construction of an intronless mouse thymidylate synthase (TS) minigene that has the normal 5' and 3' flanking regions of the gene linked to full length TS cDNA. Transfection of the minigene into ts- hamster V79 cells led to low level expression of normal mouse TS mRNA and protein. In the present study we analyzed the effect of introns on the expression of the TS minigene in transient transfection assays. Inclusion of introns 5 and 6 at their normal locations in the coding region led to an 8-9-fold stimulation of the level of TS and TS mRNA. Almost all of introns 5 and 6 could be deleted without diminishing the stimulatory effect. Inclusion of intron 3 also stimulated the expression of the minigene, although to a lesser extent than introns 5 and 6. However, inclusion of intron 4 had no stimulatory effect. Analysis of minigenes that contained various combinations of introns revealed that the stimulatory effects of the introns were not additive.     

Intron requirement for expression of the human purine nucleoside phosphorylase gene.

Abbreviated purine nucleoside phosphorylase (PNP) genes were engineered to determine the effect of introns on human PNP gene expression. PNP minigenes containing the first intron (complete or shortened from 2.9 kb down to 855 bp), the first two introns or all five PNP introns resulted in substantial human PNP isozyme expression after transient transfection of murine NIH 3T3 cells. Low level human PNP activity was observed after transfection with a PNP minigene containing the last three introns. An intronless PNP minigene construct containing the PNP cDNA fused to genomic flanking sequences resulted in undetectable human PNP activity. Heterogeneous, stable NIH 3T3 transfectants of intron-containing PNP minigenes (verified by Southern analysis), expressed high levels of PNP activity and contained appropriately processed 1.7 kb message visualized by northern analysis. Stable transfectants of the intronless PNP minigene (40-45 copies per haploid genome) contained no detectable human PNP isozyme or mRNA. Insertion of the 855 bp shortened intron 1 sequence in either orientation upstream or downstream of a chimeric PNP promoter-bacterial chloramphenicol acetyltransferase (CAT) gene resulted in a several-fold increase in CAT expression in comparison with the parental PNP-CAT construct. We conclude that human PNP gene expression at the mRNA and protein level is dependent on the presence of intronic sequences and that the level of PNP expression varies directly with the number of introns included. The disproportionately greatest effect of intron 1 can be explained by the presence of an enhancer-like element retained in the shortened 855 bp intron 1 sequence.     

Structure and expression of a rice hsp70 gene

A genomic hsp70 gene was isolated from a rice IR36 genomic library and 4 794 bp of the gene have been sequenoed. The 5' flanking region of the gene contained a putative TATA box and a typical heat shock element sequence 5'-CTcgGAAccTTCgAG-3'. The amino acid sequence of the rice HSP70 deduced from the coding region shared 84%-92% homologies with those of HSP70s from other plant species. An intron 1939bp long was identified in the coding region at the codon specifying amino acid 72 (Asp), the similar position introns occurring in other intron-containing hsp70 genes. In addition, another intron of 57 bp was found in the 3'-untranslated region in the rice hsp70 gene. Southern blot hybridization showed that rice hsp70 gene family contained at least three members. Analysis of the RNA leveis with the gene-specific and non-specific probes revealed that the rice hsp70 gene expressed at normal temperature and the expression was enhanced by heat shock treatment.     

Structure and chromosome localization of the human eosinophil-derived neurotoxin and eosinophil cationic protein genes: Evidence for intronless coding sequences in the ribonuclease gene superfamily

Human genomic DNAs for the eosinophil granule proteins, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), were isolated from genomic libraries. Alignment of EDN (RNS2) and ECP (RNS3) gene sequences demonstrated remarkable nucleotide similarities in noncoding sequences, introns, and flanking regions, as well as in the previously known coding regions. Detailed examination of the 5'-noncoding regions yielded putative TATA and CAAT boxes, as well as similarities to promoter motifs from unrelated genes. A single intron of 230 bases was found in the 5' untranslated region and we suggest that a single intron in this region and an intronless coding region are features common to many members of the RNase gene superfamily. The RNS2 and RNS3 genes were localized to the q24-q31 region of human chromosome 14. It is likely that these two genes arose as a consequence of a gene duplication event that took place approximately 25-40 million years ago and that a subset of anthropoid primates possess both of these genes or closely related genes.     

A splicing enhancer in the 3'-terminal c-H-ras exon influences mRNA abundance and transforming activity.

Analysis of cDNA clones previously identified an optional intron in the 3'-untranslated region of the human H-ras gene. A possible correlation was observed between failure to remove this intron and overexpression of the gene, suggesting that splicing of the intron may require a specific titrable factor. The splicing signals at the end of the intron deviate from the consensus and may be inefficient, but we noticed that the adjacent exon downstream has a purine-rich region reminiscent of purine-rich splicing enhancers in other genes that stimulate the removal of weak, flanking introns. We show here that the purine-rich region of H-ras has splicing-enhancer activity in the homologous as well as a heterologous context. Interestingly, although the affected intron is outside the coding region, inversion or deletion of the enhancer reduced the transforming activity of oncogenic H-ras alleles severalfold. Experiments with corresponding cDNA constructs suggested that this is not a consequence of the altered structures of the mRNAs produced when the enhancer is inverted or deleted. Instead, we propose that the region controls an additional pre-mRNA processing event besides splicing of the terminal intron. Our work indicates that the purine-rich region may play an important role in the control of H-ras activity.     

The nucleotide sequence of the chick cytoplasmic beta-actin gene

The nucleotide sequence of the chick beta-actin gene was determined. The gene contains 5 introns; 4 interrupt the translated region at codons 41/42, 120/122, 267, 327/328 and a large intron occurs in the 5' untranslated region. The gene has a 97 nucleotide 5'-untranslated region and a 594 nucleotide 3'-untranslated region. A slight heterogeneity in the position of the poly A addition site exists; polyadenylation can occur at either of two positions two nucleotides apart. The gene codes for an mRNA of 1814 or 1816 nucleotides, excluding the poly(A) tail. In contrast to the chick skeletal muscle actin gene the beta-actin gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. In the 5' flanking DNA, 15 nucleotides downstream from the CCAAT sequence, is a tract of 25 nucleotides that is highly homologous to the sequence found in the same region of the rat beta-actin gene.