An analysis of vertebrate mRNA sequences: intimations of translational control.

Kozak, Marilyn

doi:10.1083/jcb.115.4.887

Cited by 1,579 publications

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“…An expressed sequence tag (EST) for HYAL1 from neuroepithelial cells (AA223264) was found in the EST database (dbEST) that skipped nucleotides 103 ± 585, suggesting that this portion of the 5' UTR might contain a retained intron (Kozak, 1996). A retained intron can severely limit translation by preventing the ribosome from binding to the correct initiating methionine codon (Kozak, 1991). The previous primers used would not have ampli®ed such a spliced form, as the forward primer was within this potential ®rst intron.…”

Section: Sequencing Of the Full-length Cdna Fails To Identify Mutatiomentioning

confidence: 99%

HYAL1LUCA-1, a candidate tumor suppressor gene on chromosome 3p21.3, is inactivated in head and neck squamous cell carcinomas by aberrant splicing of pre-mRNA

et al. 2000

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The hyaluronidase ®rst isolated from human plasma, Hyal-1, is expressed in many somatic tissues. The Hyal-1 gene, HYAL1, also known as LUCA-1, maps to chromosome 3p21.3 within a candidate tumor suppressor gene locus de®ned by homozygous deletions and by functional tumor suppressor activity. Hemizygosity in this region occurs in many malignancies, including squamous cell carcinomas of the head and neck. We have investigated whether cell lines derived from such malignancies expressed Hyal-1 activity, using normal human keratinocytes as controls. Hyal-1 enzyme activity and protein were absent or markedly reduced in six of seven carcinoma cell lines examined. Comparative genomic and¯uorescence in situ hybridization identi®ed chromosomal deletions of one allele of HYAL1 in six of seven cell lines. Initial RT ± PCR analyses demonstrated marked discrepancies between levels of HYAL1 mRNA and protein. Despite repeated sequence analyses, no mutations were found. However, two species of transcripts were identi®ed when primers were used that included the 5' untranslated region. The predominant mRNA species did not correlate with protein translation and contained a retained intron. A second spliced form lacking this intron was found only in cell lines that produced Hyal-1 protein. Inactivation of HYAL1 in these tumor lines is a result of incomplete splicing of its pre-mRNA that appears to be epigenetic in nature.

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Section: Sequencing Of the Full-length Cdna Fails To Identify Mutatiomentioning

confidence: 99%

HYAL1LUCA-1, a candidate tumor suppressor gene on chromosome 3p21.3, is inactivated in head and neck squamous cell carcinomas by aberrant splicing of pre-mRNA

et al. 2000

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“…These seven nucleotides were kept to retain the 'start codon context'. 28 The truncated IFN-g cDNA was produced via restriction digestion of the wild-type cDNA at the HindIII and DraI restriction sites. Mutated versions of the tested cytokine genes retained their 3'UTR, but the AU-rich regions containing ATTTA pentamers were replaced by four tandem repeats of AUGUA via an overlap extension PCR cloning as described by Rajagopalan and Malter.…”

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confidence: 99%

Molecular strategies for improving cytokine transgene expression in normal and malignant tissues

Turner

2003

Gene Ther

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The augmentation and optimization of specific targeted transgene expression systems are important strategies for clinical research into gene therapy and DNA vaccination, due to safety considerations. In this study, we introduced 3 0 untranslated regions and transcriptional control modifications and direct tandem or combinational vector design strategies into a number of specific cytokine cDNA expression plasmids. The experiments were performed in parallel using both in vivo and in vitro transgene expression systems. In vivo studies were carried out using gene gun delivery of test vectors into mouse skin tissues. A combination of specific cell lines and fresh cell explants were used for in vitro and ex vivo transgene expression assay systems. The results from these comparative experiments demonstrated that a number of molecular biology manipulations can be readily adapted to define and significantly enhance the level or/and duration of transgene expression for a group of clinically relevant cytokine genes, with very similar effects for both in vivo and in vitro test systems. This cytokine transgene expression system may offer a favorable means for improving the efficiency of cytokine gene therapy and DNA vaccines in future clinical studies.

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“…In addition, a commonly used mechanism is initiation of translation from codons other than the classical AUG (e.g. ACG, CUG, or GUG) (Kozak, 1991). In preliminary experiments, our initial hypothesis that p15.5 and p15 are initiated from the two in frame AUG codons spaced six codons apart (Figure 1b) turned out to be incorrect (see below).…”

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“…ACG, CUG, or GUG) located upstream at the ®rst AUG codon. Such initiation is usually ine cient and occurs in addition to initiation at the ®rst AUG (for references, see Kozak, 1991). Due to this leakiness at the alternative initiation codon, two (or more) products are synthesized.…”

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confidence: 99%

“…One sample was used to detect the isoforms by immunoblotting (lanes 1 and 2), while the other two samples were ®rst subjected to immunoprecipitation with antisera against CDK4 (H22, Santa Cruz) or CDK6 (C21, Santa Cruz), followed by separation on a 15% SDS-gel, immunoblotting with anti-p15 antiserum (C-20, Santa Cruz) (lanes 3 ± 6), and visualization by ECL (Kozak, 1989). The same rules also apply to initiation at the alternative codons (Kozak, 1991). The context around the GUG codon initiating p15.5 synthesis (ACGGUGGAU) is very close to the Kozak consensus sequence with an A at position 73 and a G in position +4.…”

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Translation of p15.5INK4B, an N-terminally extended and fully active form of p15INK4B, is initiated from an upstream GUG codon

2000

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The expression of the cyclin-dependent kinase inhibitor p15 INK4B in normal cells after induction with TGF-b1, or following overexpression from an adenovirus-encoded cDNA, appears on an SDS-polyacrylamide gel as a doublet. Here, the underlying mechanism behind the synthesis of the two species has been studied. By expressing cDNAs truncated at their 5' end, we found that the synthesis of the more slowly migrating form, called p15.5 INK4B , is dependent on a sequence upstream of the ®rst AUG codon thought to initiate translation of p15 INK4B . Two potential, in frame, alternative upstream initiation codons, ACG and GUG, were individually changed to GCA encoding alanine. Analysis by in vitro translation, or immunoblotting of lysates from transfected 293 cells, showed that translation of p15.5 INK4B is initiated at the GUG located 13 codons upstream of the ®rst AUG. When this AUG was mutated, p15 INK4B was no longer made. Instead, a shorter form, initiated at an in frame AUG located seven codons downstream, was synthesized. Finally, when both these AUGs were mutated, only p15.5 INK4B was generated. Both p15 INK4B and p15.5 INK4B bound to CDK4 and CDK6, inhibited DNA synthesis, and caused replicative senescence of a human glioma cell line. We thus conclude that p15 INK4B and p15.5 INK4B , encoded by the CDKN2B gene, are functionally indistinguishable as based on these assays.

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An analysis of vertebrate mRNA sequences: intimations of translational control.

Cited by 1,579 publications

References 432 publications

HYAL1LUCA-1, a candidate tumor suppressor gene on chromosome 3p21.3, is inactivated in head and neck squamous cell carcinomas by aberrant splicing of pre-mRNA

HYAL1LUCA-1, a candidate tumor suppressor gene on chromosome 3p21.3, is inactivated in head and neck squamous cell carcinomas by aberrant splicing of pre-mRNA

Molecular strategies for improving cytokine transgene expression in normal and malignant tissues

Translation of p15.5INK4B, an N-terminally extended and fully active form of p15INK4B, is initiated from an upstream GUG codon

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