Single Coding Sequence Research Articles

Bicistronic expression of nuclear transgenes in Chlamydomonas reinhardtii.

In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co-expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)-α-bisabolene production of 7.95 mg L-1 from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high-level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co-expression from the algal nuclear genome.

Quadrature Phase-Shift Keying Modulation With Random Coding Pulse for Long-Range ϕ-OTDR

Transient effect in erbium-doped fiber amplifier (EDFA) restricts the application of the phase-sensitive optical time-domain reflectometry (ϕ-OTDR) system with coding probe pulse (CPP) in long-distance vibration sensing. In this paper, a CPP modulation scheme based on phase coding is proposed to suppress the amplitude distortion after optical amplification. Quadrature phase-shift keying (QPSK), which has a high spectrum utilization efficiency, is designed on a I/Q modulator. The random sequence CPP is used in this system, because of the single coding sequence, the signal-to-noise ratio (SNR) can be improved without loss of the sensing bandwidth. Experimental results prove the effectiveness of the scheme. Compared to the conventional modulation scheme at a 50 mW pump power, the distortion coefficient of the CPP after amplification decreases from 57.67% to 9.23%, and the signal-to-interference ratio (SIR) increases from 13.89 dB to16.67 dB. Without distributed amplification, the proposed system successfully located the vibration at 85.652 km under the carrier period of 40 ns, and the SNR of the demodulated signal is up to 21.99 dB.

M-ARY CDMA SCHEME WITH CCSK MODULATION AND PARALLEL INTERFERENCE CANCELLATION TECHNIQUE

In this paper, we tackle the problem of implementation a parallel interference cancellation scheme for multi-user detection (MUD) in M-ary CCSK-modulated CDMA system. Complexity remains the main obstacle to the practical implementation of MUD and a factor limiting, if not the very possibility, then the feasibility of using high order M-ary modulation formats for applications such as low-probability-of-intercept systems with a large spreading factor or, myriads of small self-powered IoT devices. The design of MUD algorithms for M-ary systems leads to a new round of growth in the receiver complexity comparing with the binary modulation. Demodulator has a major impact on the complexity of CDMA system. Cyclic code-shift keying (CCSK) is a modulation technique which is designed to reduce the complexity of M-ary signaling. In this, each symbol is a circularly shifted version of a single code sequence. Assuming synchronization, the receiver cyclically correlates the input signal plus noise with the base sequence and estimates the position of the correlation peak. The preliminary stage of the proposed scheme is a conventional multi-channel CDMA receiver. The preliminary estimates of the data is multiplied by user codes and amplitude estimates in the spreader, and then fed to the adder to generate the MAI estimates. After the latter are subtracted from the group signal, K-dimensional vector of cleared user signals is passed to the matched filter bank to form the refined estimates of the data. At each subsequent stage, the estimates of the output data of the previous stage are used as input data. The complexity gain is the ratio of the number of computations for finding cyclic convolution and, the amount of computations for finding M linear convolutions, i.e., M/log2M. Since the performance of the algorithm is sensitive to the reliability of the preliminary decisions, there are strict criteria for the selection of spreading codes: the length which must be an integer power of 2, the large family size, good periodic autocorrelation and good cross-correlation properties. To examine the proposed algorithm using computer simulation, we selected minimax periodic and odd-periodic complementary codes, the properties of which are close to the properties of the codes used in the CCSK scheme implemented in Link-16 protocol tactical data networks JTIDS and MIDS. Our study shows that the gain over the conventional receiver increases as the SNR increases, achieving 15 dB for BER 10-5. The system with complementary codes outperform system based on minimax PN-codes, achieving a target bit error rate at a lower SNR.

Death of a dogma: eukaryotic mRNAs can code for more than one protein.

mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5′ UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3′ UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma.

Genetic robustness at the codon level as a measure of selection

Selection at the DNA level is usually detected by analysing substitution rates from multiple-species comparisons. It has been suggested that measures of genetic robustness at the codon level, which can be measured by analysing a single coding sequence, can be used to estimate selection, but the validity of these measures has been questioned. Here I test the efficiency of different measures of genetic robustness at the codon level to estimate the level of selection acting on a gene. I find that volatility and other measures of robustness are correlated with dN/ dS, and that this is not simply the effect of a preference for translationally optimal codons. I discuss the possible implications and the possible problems of these methods based on single-sequence codon usage analysis.

Matching curated genome databases: a non trivial task

BackgroundCurated databases of completely sequenced genomes have been designed independently at the NCBI (RefSeq) and EBI (Genome Reviews) to cope with non-standard annotation found in the version of the sequenced genome that has been published by databanks GenBank/EMBL/DDBJ. These curation attempts were expected to review the annotations and to improve their pertinence when using them to annotate newly released genome sequences by homology to previously annotated genomes. However, we observed that such an uncoordinated effort has two unwanted consequences. First, it is not trivial to map the protein identifiers of the same sequence in both databases. Secondly, the two reannotated versions of the same genome differ at the level of their structural annotation.ResultsHere, we propose CorBank, a program devised to provide cross-referencing protein identifiers no matter what the level of identity is found between their matching sequences. Approximately 98% of the 1,983,258 amino acid sequences are matching, allowing instantaneous retrieval of their respective cross-references. CorBank further allows detecting any differences between the independently curated versions of the same genome. We found that the RefSeq and Genome Reviews versions are perfectly matching for only 50 of the 641 complete genomes we have analyzed. In all other cases there are differences occurring at the level of the coding sequence (CDS), and/or in the total number of CDS in the respective version of the same genome.CorBank is freely accessible at . The CorBank site contains also updated publication of the exhaustive results obtained by comparing RefSeq and Genome Reviews versions of each genome. Accordingly, this web site allows easy search of cross-references between RefSeq, Genome Reviews, and UniProt, for either a single CDS or a whole replicon.ConclusionCorBank is very efficient in rapid detection of the numerous differences existing between RefSeq and Genome Reviews versions of the same curated genome. Although such differences are acceptable as reflecting different views, we suggest that curators of both genome databases could help reducing further divergence by agreeing on a minimal dialogue and attempting to publish the point of view of the other database whenever it is technically possible.

A Simplified Single Correlator Rake Receiver for CDMA Communications

This paper presents a single correlator RAKE receiver for direct sequence code division multiple access (DS-CDMA) sys- tems. In conventional RAKE receivers, multiple correlators are used to despread the multipath signals and then to align and combine those signals in a later stage before making a bit decision. The simplified receiver structure presented here uses a single correlator and single code sequence generator to recover the multipaths. Modified Walsh- Hadamard codes are used here for data spreading that provides better uncorrelation properties for the multipath signals. The main advantage of this receiver structure is that it requires only a single correlator and a code generator in contrary to the conventional RAKE receiver concept with multiple correlators. It is shown in results that the proposed receiver achieves better bit error rates in comparison with the conventional one for more than one multipaths.

Tenebrio molitor antifreeze protein gene identification and regulation

The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

DNA vaccines prime CD8+ T cell responses to epitopes of viral antigens produced from overlapping reading frames of a single coding sequence

A hepatitis B virus (HBV)-derived sequence that encodes the 832-residue polymerase (Pol) protein of HBV in the primary open reading frame (ORF), and the three (large, middle and small) hepatitis B surface antigen (HBsAg) variants in an alternative ORF was used. This sequence was cloned into expression vectors in which Pol was expressed under heterologous (HCMV, SV40 or metallothionin) promoter control. Some Pol-encoding vectors coexpressed Pol as well as readily detectable amounts of HBsAg. Efficient HBsAg expression depended on endogenous HBV promoter sequences but was apparently also facilitated by heterologous promoter sequences located upstream of the HBV Pol sequence. DNA immunization of mice efficiently coprimed CD8(+) T cell responses to epitopes of Pol and HBsAg. Over expression of Pol (using an hsp73-facilitated expression system) did not correlate with the immunogenicity of the K(d)/Pol(140-148) epitope. Immunodominant L(d)-restricted CD8(+) T cell responses to HBsAg down-modulated priming of CD8(+) T cell responses to other HBsAg epitopes but not to the K(d)/Pol(140-148) epitope. Different antigens transcribed from alternative reading frames of a single sequence in a DNA vaccine can thus efficiently prime multispecific T cell responses.

Alternative splicing, the gene concept, and evolution

Alternative splicing allows for the production of many gene products from a single coding sequence. I introduce the concept of alternative splicing via some examples. I then discuss some current hypotheses about the explanatory role of alternative splicing, including the claim that splicing is a significant contributor to the difference in complexity between the human genome and proteosome. Hypotheses such as these bring into question our working concepts of the gene. I examine several gene concepts introduced to cope with processes such as alternative splicing. Next I introduce some hypotheses about the evolution of mechanisms alternative splicing in higher organisms. I conclude that attention to alternative splicing reveals that we adopt an attitude that developmental theorizing must inform evolutionary theorizing and vice versa.

Alternative splicing and genome complexity.

Alternative splicing of mRNA allows many gene products with different functions to be produced from a single coding sequence. It has recently been proposed as a mechanism by which higher-order diversity is generated. Here we show, using large-scale expressed sequence tag (EST) analysis, that among seven different eukaryotes the amount of alternative splicing is comparable, with no large differences between humans and other animals.

Two distinct TATA-less promoters direct tissue-specific expression of the rat apo-B editing catalytic polypeptide 1 gene.

The species and tissue specificity of apolipoprotein (apo) B mRNA editing is determined by the expression of apoB editing catalytic polypeptide 1 (APOBEC-1), the cytidine deaminase that catalyzes apoB mRNA editing. To understand the molecular mechanisms that regulate the transcription of APOBEC-1, we characterized rat APOBEC-1 cDNA and genomic DNA. cDNA cloning and RNase protection analysis showed two alternative promoters for the tissue-specific expression of APOBEC-1 in the liver and intestine, Pliv and Pint. Both promoters lack a TATA box, and Pint belongs to the MED-1 class of promoters, which initiate transcription at multiple sites. We also identified two allelic forms of the APOBEC-1 gene from the characterization of two rat APOBEC-1 P1 genomic clones, RE4 and RE5. The RE4 allele is 18 kilobases long and contains six exons and five introns, whereas the RE5 allele contains an additional approximately 8 kilobases of intron sequences and an extra exon encoding a 5'-untranslated region; however, the APOBEC-1 transcripts from the two alleles appear to have similar, if not identical, functions. Transgenic mouse studies showed that Pliv was preferentially used in the liver, kidney, brain, and adipose tissues, whereas Pint was preferentially used in the small intestine, stomach, and lung. Our results suggest that the tissue-specific expression of APOBEC-1 is governed by multiple regulatory elements exerting control over a single coding sequence. The presence or absence of these regulatory elements may determine the tissue-specific expression of APOBEC-1 in other mammalian species.

Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene.

The agouti coat color gene encodes a paracrine signaling molecule that controls the production of yellow and black pigment by melanocytes within hair follicles. Some agouti alleles affect the dorsum and ventrum independently, which has provided the basis for speculation that agouti gene action in different regions of the body is controlled by distinct genetic loci that are closely linked. Using a combination of cDNA cloning and RNA expression studies, we find that alternative isoforms of agouti mRNA contain different noncoding first exons located 100 kb apart, whose patterns of expression indicate independent control by regulatory elements that are either ventral specific or hair cycle specific. These results demonstrate that the apparent genetic complexity of the agouti locus is explained by the existence of multiple regulatory elements exerting control over a single coding sequence and provide a conceptual basis for understanding differences in dorsal and ventral hair coloration in many mammalian species. The ventral-specific agouti isoform represents an example of a transcript whose expression is restricted to ventral skin and provide an approach to investigate the mechanisms by which dorsal-ventral differences in gene expression are established and maintained.

Time, date, and event code generator to identify physiologic events simultaneously recorded on magnetic tape and oscillographic paper.

This instrument will generate upon command any one of three serial code sequences in a binary coded decimal (BCD) format representing either 1) time of day, 2) time and date, or 3) event number. Either a single code sequence or continuously repeating sequences can be generated. The instrument is intended to be used in a biomedical data acquisition system to permanently identify, index, and synchronize analog or digital data recorded on magnetic tape or oscillographic paper. Three-digit event codes, consecutively numbered 1 through 999 in successive frames, are particularly valuable for indexing experimental maneuvers or deliberate changes in experimental variables. Individual digits in the code format are easier to interpret visually than are corresponding digits in recognized time code formats.