Terminal Coding Sequence Research Articles

Codon usage bias in 5′ terminal coding sequences reveals distinct enrichment of gene functions

Codon bias at the 5′ terminal of coding sequence (CDS) is known to be distinct from the rest of the CDS. A number of events occur in this short region to regulate early translation elongation and co-translational translocation. In the genes encoding secretory proteins, there is a special signal sequence which has a higher occurrence of rare codons. In this study, we analyzed codon bias of secretory genes in several eukaryotes. The results showed that secretory genes in the species except mammals had a higher occurrence of rare codons in the 5′ terminal of CDS, and the bias was greater than the same region of non-secretory genes. GO analysis revealed that secretory genes containing rare codon clusters in different regions were responsible for various roles in gene functions. Moreover, codon bias in the region encoding the hydrophobic region of protein is similar in secretory and non-secretory genes, indicating that codon bias in secretory genes was partly influenced by amino acid bias. Rare codon clusters are found more frequently in specific regions, and continuous rare codons are not favoured probably because they will increase the probability of ribosome collision and drop-off. Based on ribosome profiling data, there is no significant difference in the average translation efficiencies between rare and optimal codons. Higher ribosomal density in the 5′ terminal may result from ribosome pausing which could be involved in different translation events. These findings collectively provided rich information on codon bias in secretory genes, which may shed light on the co-effect of codon bias, mRNA structure and tRNA abundance in translational regulations.

Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor.

The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and -SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3' terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane.

Identification of two functionally redundant RNA elements in the coding sequence of poliovirus using computer-generated design

Genomes of RNA viruses contain multiple functional RNA elements required for translation or RNA replication. We use unique approaches to identify functional RNA elements in the coding sequence of poliovirus (PV), a plus strand RNA virus. The general method is to recode large segments of the genome using synonymous codons, such that protein sequences, codon use, and codon pair bias are conserved but the nucleic acid sequence is changed. Such recoding does not affect the growth of PV unless it destroys the sequence/structure of a functional RNA element. Using genetic analyses and a method called “signal location search,” we detected two unique functionally redundant RNA elements (α and β), each about 75 nt long and separated by 150 nt, in the 3′-terminal coding sequence of RNA polymerase, 3D pol . The presence of wild type (WT) α or β was sufficient for the optimal growth of PV, but the alteration of both segments in the same virus yielded very low titers and tiny plaques. The nucleotide sequences and predicted RNA structures of α and β have no apparent resemblance to each other. In α, we narrowed down the functional domain to a 48-nt-long, highly conserved segment. The primary determinant of function in β is a stable and highly conserved hairpin. Reporter constructs showed that the α- and β-segments are required for RNA replication. Recoding offers a unique and effective method to search for unknown functional RNA elements in coding sequences of RNA viruses, particularly if the signals are redundant in function.

Identification and Elimination of an Aberrant Splice Product from cDNAs Encoding the Human Adenovirus Type 5 E4orf6 Protein

Growing awareness of the central role of the E4orf6 protein in regulating the infectious cycle of human adenoviruses has led to greatly intensified efforts to define its functions and mechanisms of action. Many workers employ cDNAs to generate plasmid or viral vectors to express E4orf6 in the absence of other viral products. In addition to the normal 34-kDa product, such vectors consistently produce a polypeptide of about 8 kDa. In the present report we show that this protein is produced by an aberrant mRNA utilizing the 5′ splice donor site used normally by the virus to produce the E4orf6/7 product, which shares 58 residues with E4orf6. This amino terminal coding sequence is linked to a 3′ sequence via a novel splice acceptor site in an alternative reading frame of the E4orf6 cDNA. The 5′ donor site was altered by PCR-directed mutagenesis to yield a construct that produces high levels of E4orf6 in the absence of the 8-kDa polypeptide. This construct should eliminate some of the problems encountered previously using the wild-type E4orf6 coding sequence.

Transactivation of Dianthin Transgene Expression by African Cassava Mosaic Virus AC2

We have recently described a novel strategy for engineering resistance to African cassava mosaic virus (ACMV) in transgenicNicotiana benthamianaplants using a virus-inducible promoter to control the expression of a plant ribosome-inactivating protein (RIP) transgene (Y. Honget al., Virology220, 119–127, 1996). Here, we have used a potato virus X (PVX) vector to express the ACMV transactivator protein, AC2,in planta.We confirm that amplification of RIP activity in transgenic plants is mediated by AC2; disruption of AC2 expression by either the introduction of an in-frame stop codon or the deletion of 5′-terminal or 3′-terminal coding sequences reduced RIP expression to the basal level associated with PVX-infected plants. AC2 expression from the PVX vector induced necrosis in nontransformed plants as well as in plants containing the RIP transgene, suggesting that the protein can functionally interact with PVX and/or host factors. The potential of this system to provide a direct and sensitive assay to investigate AC2 functionin plantais discussed.

Structure and analysis of the mouse Na+/H+ exchanger (NHE1) gene: homology and conservation of splice sites.

The Na+/H+ exchanger is a widely distributed integral membrane protein that is responsible for pH regulation in mammalian tissues. We have cloned and analyzed the NHE1 isoform of the mouse genomic Na+/H+ exchanger. A clone from a mouse genomic library contained the NHE1 promoter region and the 5'-untranslated region. It also contained the first 121 amino acids of the coding region of the Na+/H+ exchanger. A splice site occurred after amino acid 121, at the same region as in the human NHE1 gene. The deduced amino terminal coding sequence was 76 and 88% identical to the human and rat NHE1 sequences respectively. The 5'-untranslated region was highly homologous to that of other species and two minicistrons contained in the human Na+/H+ exchanger were present in the mouse sequence. The results show that the deduced protein sequence of the mouse NHE1 gene has a high level of homology with other species and that the splice site of the first intron is conserved. These results suggest that the first large intron may play an important role in the NHE1 gene expression.

V(D)J recombination coding junction formation without DNA homology: processing of coding termini.

Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining.

V(D)J Recombination Coding Junction Formation without DNA Homology: Processing of Coding Termini

Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining.

Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (Polyhedrin gene) region

The nucleotide sequence of a 9.4-kbp region including the polyhedrin gene of the C6 strain of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome was determined. These data provide a complete description of the EcoRI-I fragment, which is used to produce transfer vectors for inserting foreign genes into the AcMNPV. Ten potential open reading frames (ORFs) were identified in the complete sequence, on either strand of DNA. The largest of these was 1629 nucleotides in length and was located downstream from the polyhedrin coding sequences, but on the opposite strand of DNA. Northern blot hybridization analysis of ORF 8 (1629) identified an RNA of 2000 nucleotides which was produced in infected cells from 12 hr p.i. and remained until at least 48 hr p.i. S1 nuclease mapping and analysis of cDNA clones located the 3′ end of the mRNA at a site 16 nucleotides downstream of the polyhedrin coding sequences. The 5′ end of the mRNA was mapped using primer extension analysis of polyadenylated RNA. ThemRNA start site was positioned within a late/very late consensus transcription initiation motif (ATAAG), 428 nucleotides upstream from the potential ATG translation initiation codon. The biological significance of the putative gene product was assessed by inserting a synthetic oligonucleotide in the carboxyl terminal coding sequences of ORF 8 (1629) to prematurely terminate translation. Recombinant viruses containing this mutation were not isolated, suggesting that the ORF 1629 gene product is essential for virus replication.

Isolation and sequence of a cDNA encoding human platelet phosphofructokinase

A cDNA encoding human platelet 6-phosphofructokinase (PFK; EC 2.7.1.11) has been isolated from a human lymphocyte Raji cell line cDNA library using a cDNA for human muscle PFK as a probe. The platelet cDNA contains 900bp of carboxy terminal coding sequence and 238bp of downstream untranslated region. The deduced amino acid sequence shows 71% identity to the amino acid sequence for the human muscle isoenzyme and 63% identity to the human liver isoenzyme. Almost all of the amino acid residues contributing to catalytic and effector sites in the three isoenzymes are conserved. The platelet gene has been assigned to chromosome 10p15.2-p15.3 by using the cDNA clone as a biotinylated probe against human chromosome spreads (Morrison et al. 1991, submitted to Human Genetics).

Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II

Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E. coli-induced disease. SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells. In this study, functional boundaries of the slt-II operon were mapped. Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity. However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically. Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin. Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit. Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.

Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA.

Adenovirus DNA is tightly bound to the nuclear matrix throughout the course of infection. Analysis of adenovirus DNA from infected HeLa cell nuclei after extraction with lithium diiodosalicylate and digestion with restriction enzymes demonstrated that the sites of tightest attachment occur in the terminal fragments of the linear viral chromosome. Analysis of viruses mutated in the precursor terminal protein coding sequence demonstrated that the terminal protein, which is covalently attached to the 5' end of each DNA strand, mediates the tight binding. Virions containing chromosomes with mutant terminal proteins were unpackaged and viral DNA accumulated in the nucleus at a normal rate and competed for the limiting component during transcription complex formation, but their early genes were transcribed at reduced efficiency by both RNA polymerases II and III. The transcriptional defects were not complemented by coinfection with a wild-type virus. We propose that the adenovirus chromosome may exist as a single chromatin domain during infection and that binding of DNA to the nuclear matrix may play a critical role in adenovirus transcription.

Expression of the oxi1 and maturase-related RF1 genes in yeast mitochondria.

Transcription of the yeast mitochondrial oxi1 gene (cytochrome oxidase subunit 2) is initiated at a variant non anucleotide sequence, TTAAAAGTA, located 54 bp upstream from the protein-coding gene. Transcriptional initiation at this site gives rise to a 2,500 nucleotide primary transcript containing both the oxi1 gene and the downstream maturase-related reading frame, RF1. Precise transcript mapping has revealed that the 3'-end of the mature oxi1 mRNA is generated by an endonucleolytic cleavage which takes place after the conserved dodecamer sequence, AAUAAUAUUCUU (End-of-Messenger signal), 75 nucleotides downstream from the oxi1 stop codon. Since the RF1 5'-terminal coding region overlaps the oxi1 3'-terminal coding sequence, cleavage at this motif truncates the RF1 message suggesting that the expression of the putative RF1 protein is controlled at the level of dodecamer processing.

Identification of the genes in the Escherichia coli ileS-lsp operon. Analysis of multiple polycistronic mRNAs made in vivo.

The genes encoding isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) of Escherichia coli were previously shown to be co-transcribed (Miller, K. W., and Wu, H. C. (1987) J. Biol. Chem. 262, 389-393). However, the boundaries of this transcriptional unit have not been established. In this regard, DNA sequence determination has shown that ileS and lsp are closely flanked by four open reading frames, i.e. x-ileS-lsp-orf149-orf316-orf304. To define the boundaries of the operon, we applied Northern blotting hybridization and mRNA 5'-end mapping to analyze mRNA from a wild-type strain (SM31) and a mutant strain (SM31-2B4) that exhibits an increased expression of prolipoprotein signal peptidase. Four ileS-lsp co-transcripts were detected in RNA from the strain SM31. In addition to these four mRNAs, two new, highly abundant co-transcripts were also detected in RNA from the mutant. Based upon the determination of the 5'-ends of the mRNAs and analysis of their coding sequences, we conclude that the six mRNAs actually are comprised of three pairs of related mRNAs. The two mRNAs in a given pair have the same 5'-termini (all located upstream of or within gene x), but vary with respect to the identity of their 3'-terminal coding sequence (lsp or orf316). In conclusion, the ileS-lsp operon contains five genes, x-ileS-lsp-orf149-orf316, whose transcription probably is dependent upon promoter(s) located upstream of or within gene x. The next gene downstream, orf304, apparently does not reside in the operon.

Juvenile hormone-regulated locust vitellogenin genes: Lack of expression after transfer intoDrosophila

In Locusta migratoria, vitellogenin (Vg) is normally produced only in adult female fat body under stimulation by juvenile hormone (JH). This permits study of (a) programming of genes for expression and (b) modulation of expression by JH. From L. migratoria genomic libraries, two Vg genes (A and B) have been cloned. Coding regions encompass 10-12 kbases of DNA, contain introns, and hybridize with 6,300 nucleotide mRNAs. Although the 5'-exons, coding for signal peptides, are similar in sequence, the remaining coding sequences have distinct restriction maps and show no crosshybridization. Upstream DNA from the two genes has some sequence similarity, corresponding to potential regulatory regions. Dot hybridization assays of mRNAs A and B in locust fat body after induction by methoprene show coordinate expression of the two Vg genes and also a third, unidentified JH-regulated gene. In the hope of providing a system for identification of control sequences, P element-mediated transformation has been used to transfer locust DNA into Drosophila. From Vg gene B, a central block was deleted, to give a mini-Vg gene comprising 5' and 3' terminal coding sequences plus upstream flanking DNA. This was incorporated into the P element vector Carnegie 20 and injected into Drosophila embryos. Three transformed fly lines were obtained in which the locust mini-Vg gene was integrated at different chromosomal sites. On Northern blots of fly RNA, however, no expression of the locust sequences could be detected, even though the accompanying rosy gene was expressed. This suggests that the locust regulatory sequences were not recognized in Drosophila, and current effort is directed toward developing a homologous locust transformation system for expression assay of cloned JH-regulated genes.

Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions.

A chromosomal segment with a unique structure around the immunoglobulin heavy chain joining region (JH) has been molecularly cloned from an Abelson murine leukemia virus-transformed cell line. Attached to JH3 in the cloned DNA, in inverted sequence, is the DNA from JH1 to the JH2 recognition sequence. The inverted segment is attached at its other end to the 5' recognition sequence of a diversity segment (D). To form this structure, three joining events must have occurred on the same chromosome. One of these events could have been a normal D-JH joining but the others must have been irregular events including ones that result in inversions. One of the joining events left fused recognition elements from JH2 and a D whose sequence shows that, during joining, reciprocal joinings of the recognition elements must occur to fuse the heptameric elements back to back. Because joined D and JH undergo deletion of terminal coding sequence during recombination but the joined heptameric recognition sequences do not contain the deleted sequence, joining must be a nonreciprocal event. Also, extra nucleotides are inserted between D and JH as part of the joining process; it is suggested that this added sequence is a product of the activity of terminal deoxynucleotidyltransferase at the D/JH (and probably the VH/D) joints and that it represents a new element of heavy chain gene structure, the N region.

The structure of the yeast ribosomal RNA genes. 2. The nucleotide sequence of the initiation site for ribosomal RNA transcription.

The 5'-terminal coding sequence for the 37 S precursor to rRNA of Saccharomyces cerevisiae is identified by reverse transcriptase extension and protection mapping with nuclease S1. The sequence of a 419 bp rDNA fragment containing the transcription initiation site and its adjacent region is determined.

The putative promoter of a Xenopus laevis ribosomal gene is reduplicated.

With the aid of a novel poly-dA tailing-partial restriction technique and S1-protection mapping, the 5' terminal coding sequence for the 40S precursor ribosomal RNA of Xenopus laevis has been exactly identified. Since the promoter sequence for the 40S RNA should lie close to its 5' terminal coding sequence, we are able to conclude that the "Bam-Island" sequence reduplication (1) almost certainly represents a promoter reduplication.