The nucleotide sequence of the PRI1 gene related to DNA primase in Saccharomyces cerevisiae.

The PRI1 gene of Saccharomyces cerevisiae encodes for the p48 polypeptide of DNA primase. We have determined the nucleotide sequence of a 1,965 bp DNA fragment containing the PRI1 locus. The entire coding sequence of the gene lies within an open reading frame, and there are 409 amino acids in the single polypeptide protein if translation is assumed to start at the first ATG in this frame. The 5' and 3' end-points of PRI1 mRNA have been determined by S1 mapping and primer extension analysis. The primary structure and the codon usage of PRI1 suggest that this essential gene is poorly expressed in yeast cells.     

Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency

Precise prediction of prokaryotic translation efficiency can provide valuable information for optimizing bacterial host for the production of biochemical compounds or recombinant proteins. However, dynamic changes in mRNA folding throughout translation make it difficult to assess translation efficiency. Here, we systematically determined the universal folding regions that significantly affect the efficiency of translation in Escherichia coli. By assessing the specific regions for mRNA folding, we could construct a predictive design method, UTR Designer, and demonstrate that proper codon optimization around the 5′-proximal coding sequence is necessary to achieve a broad range of expression levels. Finally, we applied our method to control the threshold value of input signals switching on a genetic circuit. This should increase our understanding of the processes underlying gene expression and provide an efficient design principle for optimizing various biological systems, thereby facilitating future efforts in metabolic engineering and synthetic biology.     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

Characterization and quantitation of myosin heavy-chain mRNA from embryonic cardiac tissue

The messenger RNA (mRNA) coding for myosin heavy chain from the 16-day-old chick embryonic cardiac tissue was purified by a rapid isolation procedure and characterized. The mRNA can be translated with fidelity under optimally chosen conditions. The protein synthesized in response to the RNA was a polypeptide of 200,000 molecular weight, identical to the authentic myosin heavy chain from the homologous chick heart tissue. The purity of the mRNA was assessed by electrophoresis in denaturing gels, by immunoprecipitation of the translation product, and by analysis of the kinetics of hybridization with the complementary DNA (cDNA). The cDNA reassociated with myosin heavy-chain mRNA with kinetics characteristic of a pure mRNA. The sequence complexity data indicated that in the 16-day-old chick embryonic heart cells there is a single mRNA sequence coding for myosin heavy chain in contrast to two different mRNA sequences reportedly present in the skeletal muscle cells (M. Patrinou-Georgoulas and H. A. John, 1977, Cell12, 491).     

Translation attenuation regulation of chloramphenicol resistance in bacteria — a review

The chloramphenicol (Cm)-inducible cat and cmlA genes are regulated by translation attenuation, a regulatory device that modulates mRNA translation. In this form of gene regulation, translation of the Cm^R coding sequence is prevented by mRNA secondary structure that sequesters its ribosome-binding site (RBS). A translated leader of nine codons precedes the secondary structure, and induction results when a ribosome becomes stalled at a specific site in the leader. Here we demonstrate that the site of ribosome stalling in the leader is selected by a cis effect of the nascent leader peptide on its translating ribosome.     

The 5′-untranslated region of the maize alcohol dehydrogenase gene provides efficient translation of mRNA in plants under stress conditions

The reduced level of expression of most cell proteins under stress conditions is determined by the low efficiency of cap-dependent translation of corresponding mRNAs. The maize gene encoding alcohol dehydrogenase, adh1, is a gene whose mRNA is efficiently translated in hypoxia. The reporter gene assay showed that the leader sequence of the adh1 mRNA provided for efficient translation of the reporter gfp gene in Nicotiana benthamiana cells in hypoxia or heat shock. The presence of this sequence in the 5′-UTR of mRNA did not change the level of expression under aerobic conditions, but the levels of gfp expression in hypoxia or heat shock were reduced five-to tenfold in the absence of this leader and remained unaffected when the adh leader sequence was present in the 5′-UTR. The adh1 leader sequence did not change the mRNA stability nor exhibited a promoter activity. Thus, the adh leader sequence acted as a translational enhancer, providing for efficient mRNA translation in plant cells under stress conditions. Introduction of this sequence into standard expression cassettes was proposed for the development of new systems to efficiently express the target proteins in plants under stress conditions.     

Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-of-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.     

Nucleotide sequence of ompV, the gene for a major Vibrio cholerae outer membrane protein

Summary The nucleotide sequence of the ompV gene of Vibrio cholerae was determined. The product of the gene is a 28,000 dalton protein which, after the removal of a 19 amino acid signal sequence, produces a mature outer membrane protein of 26,000 daltons. The cleavage site was determined by amino-terminal amino acid sequencing of the purified mature protein. The DNA upstream of the gene shows the presence of a typical promoter region as judged from the Escherichia coli consensus information; however, the Shine-Dalgarno sequence is associated with a region capable of forming a secondary structure in the mRNA. The formation of this structure would inhibit binding of the mRNA to the ribosome and reduce translation. It is proposed that this structure is recognized by a positive activator in V. cholerae and because of its absence in E. coli ompV is poorly expressed. The distribution of rare codons within ompV suggests that they may serve to slow down the translation of particular domains such that the nascent polypeptide has an opportunity to take up its conformation without interference from the later formed regions. Such a mechanism could aid localization of the protein if export were by a cotranslational secretion system.     

A 3'' co-terminal family of mRNAs from the herpes simplex virus type 1 short region: two overlapping reading frames encode unrelated polypeptide one of which has highly reiterated amino acid sequence.

We have used DNA sequencing, mRNA mapping and in vitro translation to characterise three partially overlapping genes in the genome of herpes simplex virus (HSV) type 1. These genes specify three mRNAs with distinct 5' termini but a common 3' terminus, the longest of which is immediate-early (IE) mRNA-5. The 12,000 MW (12K) IE polypeptide encoded by IEmRNA-5 is translated from an 88 codon open reading frame, leaving a 1200 base 3' non-translated region. The second mRNA (mRNA-B) is initiated within the coding sequence of IEmRNA-5, and encodes a 21K polypeptide. The 12K and 21K polypeptide coding regions do not overlap. The third mRNA (mRNA-C) is initiated within the coding region of mRNA-B, and encodes a 33K polypeptide. The reading frame for 33K has a 110 codon out-of-frame overlap with the 21K reading frame. This is the first instance of overlapping genes described for HSV. The 21K polypeptide is thought to be a DNA binding protein and is remarkable for an array of 24 tandem repeats of the sequence X/Pro/Arg (where X represents predominantly Glu, Asp, Thr, Ser or Val) in its C-terminal portion. This array, which occupies most of the region of overlap with 33K, can vary in repeat number between virus strains.     

Strict co-linearity of genetic and protein folding domains in an intragenically duplicated rat lens γ-crystallin gene

Two complementary DNA clones pRLγ-2 and pRLγ-3 of different rat lens γ-crystallin messenger RNAs have been used to identify γ-crystallin gene sequences in rat genomic DNA. Subsequently, the DNA present in the 18,000 to 20,000 bases region of the EcoRI digest, giving rise to a strong doublet hybridization signal, was cloned in λ phage Charon-4A. One of the clones, λRCHγ-3, carrying an insert of 17,500 bases has been characterized in detail. From analysis at the restriction enzyme level with 5′-, “middle” and 3′-specific subprobes of pRLγ-3 it could be deduced that λRCHγ-3 contains only one γ-crystallin gene. The coding sequences of this gene are interrupted by intronic DNA. The primary structure of this gene and its flanking regions have been established by sequencing the relevant regions of a subclone of λRCHγ-3, designated pRCHγ-3.1. The sequence data show that the γ-crystallin gene extends over 2700 bases of rat genomic DNA. The gene is split by two introns, one of 87 base-pairs after the third translation codon and a large one of 1880 base-pairs after codon 84. The mosaic structure of the gene is strictly co-linear with the structure of the γ-crystallin polypeptide in that the large intron is positioned in a region which specifies the so-called “connecting peptide” and which links the two highly symmetrical and homologous protein domains. Although expected from the cDNA and protein sequence no introns were observed between the coding regions in the DNA specifying the two homologous folding motifs present in each protein domain. The relevance of this phenomenon in terms of the evolution of the mature γ-crystallin gene is discussed.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.     

Bovine β-crystallin complementary DNA clones: Alternating proline/alanine sequence of βB1 subunit originates from a repetitive DNA sequence

A library of recombinant plasmids carrying complementary DNA sequences synthesized from bovine lens messenger RNAs was constructed. Clones coding for five different β-crystallin subunits: βB1, βB3, βBp, βs, βA3 (and βA1), were identified by means of hybridization selection, followed by one- and two-dimensional gel electrophoresis of the translational products. Under rather stringent conditions each of these clones hybridizes with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for other β-crystallins, except in the case of the homologous βA3 and βA1-crystalline. The βA3 and βA1 subunits seem to be encoded by one mRNA using two different AUG codons as start position for translation. We have also determined the nucleotide sequence of a βB1-crystallin cDNA (pBLβB1) which enabled us to deduce the complete amino acid sequence of the protein. The βB1-crystallin, a characteristic component of the high molecular weight crystallin aggregate (β_H), is internally homologous both at DNA and protein level as has been reported for γ-and other β-crystallins. This is in agreement with the idea that these proteins had a common ancestral precursor gene that internally duplicated. The G + C content of the coding sequence of βB1 is very high: 67% overall and even 84.2% for the first 170 nucleotides, due to a remarkable non-random codon usage. A proline/ alanine repetition in the N-terminal domain of the protein is encoded by a repetitive “simple” DNA sequence.