Transcriptional Regulatory Sequences Research Articles

Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutations.

Nucleotide sequencing of a 1,300-base-pair vaccinia virus DNA segment previously shown to contain a thymidine kinase (TK) gene revealed an uninterrupted reading frame of 177 codons capable of producing a polypeptide with a molecular weight of 20,102. Mapping of the TK mRNA by primer extension indicated a unique 5' end that precedes the initiation codon by only six nucleotides. Multiple 3' ends within a 10-nucleotide region, about 30 nucleotides beyond the termination codon, were located by nuclease digestion of DNA-RNA hybrids, and the length of the TK transcript, exclusive of polyadenylate, was estimated to be approximately 570 nucleotides. The region preceding the TK mRNA start site is extremely A + T rich and has sequence homologies with three other early genes. Genetic information is so compressed in this region of the DNA that the putative transcriptional regulatory sequence of the TK gene overlaps the coding sequence of a late gene. Only nine nucleotides separate the termination codon of the late gene from the initiation codon of the TK gene. Downstream, 66 nucleotides separate the TK termination codon from the apparent initiation codon of another early gene. The nature of three independent TK- mutants was revealed by nucleotide sequencing. Each has a nucleotide reiteration leading to a +1 frameshift and a nonsense codon downstream. The location of one frameshift mutation provided evidence that the first ATG is used for initiation of protein synthesis.

Vaccinia virus: a selectable eukaryotic cloning and expression vector.

Foreign DNA was inserted into two nonessential regions of the vaccinia virus genome by homologous recombination in cells infected with virus and transfected with plasmids containing the foreign DNA elements flanked by vaccinia virus DNA. Thymidine kinase-negative (TK-) recombinants were selected after inserting foreign DNA into the coding region of the TK gene of wild-type vaccinia virus; TK+ recombinants were selected after inserting the herpesvirus TK gene into TK- mutants of vaccinia virus. For TK+ expression, it was necessary to insert a 275-base-pair DNA fragment containing the initiation site and sequences upstream of an early vaccinia virus transcript next to the coding sequences of the herpesvirus gene. The unique ability of the herpesvirus TK to phosphorylate 125I-labeled deoxycytidine provided independent confirmation of gene expression. These studies demonstrate the use of vaccinia virus as a selectable cloning and expression vector, confirm the map location of the vaccinia virus TK gene, and provide initial information regarding the location of vaccinia virus transcriptional regulatory sequences.

Nucleotide sequences of the murine retrovirus Friend SFFVp long terminal repeats: identification of a structure with extensive dyad symmetry 5' to the TATA box.

The two long terminal repeats (LTRs) of the integrated provirus of the polycythemia strain of the Friend spleen focus forming virus have been sequenced. Each of the identical LTRs is 514 nucleotides long and together confer on the provirus the features of a transposable element. They are terminated by perfect 11 base-pair inverted repeats, and the entire provirus is flanked by an apparent duplication of host DNA four nucleotides long. The assumed transcription regulatory sequences (Hogness-Goldberg box, CAAT box, polyadenylation signal) can be identified within the LTRs, as well as a region of an imperfect inverted repeat which extends from approximately 140 to 270 nucleotides 5' from the point of transcription initiation. This sequence may form a hairpin-type structure which might have some function in the promotion of transcription.

Disease Education by the B.B.C

To date, the SARS-CoV-2 genome has been considered genetically more stable than SARS-CoV or MERS-CoV. Here we report a 382-nt deletion covering almost the entire open reading frame 8 (ORF8) of SARS-CoV-2 obtained from eight hospitalized patients in Singapore. The deletion also removes the ORF8 transcription-regulatory sequence (TRS), which in turn enhances the downstream transcription of the N gene. We also found that viruses with the deletion have been circulating for at least four weeks. During the SARS-CoV outbreak in 2003, a number of genetic variants were observed in the human population [1], and similar variation has since been observed across SARS-related CoVs in humans and bats. Overwhelmingly these viruses had mutations or deletions in ORF8, that have been associated with reduced replicative fitness of the virus [2]. This is also consistent with the observation that towards the end of the outbreak sequences obtained from human SARS cases possessed an ORF8 deletion that may be associated with host adaptation [1]. We therefore hypothesise that the major deletion revealed in this study may lead to an attenuated phenotype of SARS-CoV-2.