http://biology.hunter.cuny.edu/molecularbio/Class%20Materials%20Spring%202012%20Biol302/Lecture%203/Kozak%20Test%20of%20rules.pdf

Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.

The RefSeq project at the National Center for Biotechnology Information (NCBI) maintains and curates a publicly available database of annotated genomic, transcript, and protein sequence records (http://www.ncbi.nlm.nih.gov/refseq/). The RefSeq project leverages the data submitted to the International Nucleotide Sequence Database Collaboration (INSDC) against a combination of computation, manual curation, and collaboration to produce a standard set of stable, non-redundant reference sequences. The RefSeq project augments these reference sequences with current knowledge including publications, functional features and informative nomenclature. The database currently represents sequences from more than 55,000 organisms (>4800 viruses, >40,000 prokaryotes and >10,000 eukaryotes; RefSeq release 71), ranging from a single record to complete genomes. This paper summarizes the current status of the viral, prokaryotic, and eukaryotic branches of the RefSeq project, reports on improvements to data access and details efforts to further expand the taxonomic representation of the collection. We also highlight diverse functional curation initiatives that support multiple uses of RefSeq data including taxonomic validation, genome annotation, comparative genomics, and clinical testing. We summarize our approach to utilizing available RNA-Seq and other data types in our manual curation process for vertebrate, plant, and other species, and describe a new direction for prokaryotic genomes and protein name management.

Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

/pdf/interaction-of-estrogenic-chemicals-and-phytoestrogens-with-j0qbzn4u7e.pdf

Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.

/pdf/the-scanning-model-for-translation-an-update-52r2in8l71.pdf

The scanning model for translation: an update.

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.

The International Committee on Taxonomy of Viruses

We describe the current status and potential use of baculovirus vectors for the expression of foreign genes in insect cells. Trends in the development of transfer vectors for the expression of foreign genes under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus and strategies for maximizing levels of expression are discussed. Baculovirus vectors have achieved widespread acceptance for their ability to express proteins of agricultural and medical importance. A baculovirus vector was used to express the first recombinant HIV envelope proteins to receive F.D.A. approval for clinical evaluation as a candidate vaccine for AIDS. These insect DNA virus vectors are contributing to understanding the molecular biology of gene and protein function and regulation in both vertebrate and insect systems.

Trends in the development of baculovirus expression vectors

Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human beta interferon. By using specially constructed plasmids, the protein-coding sequences for interferon were linked to the AcNPV promoter for the gene encoding for polyhedrin, the major occlusion protein. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals, and the interferon-polyhedrin hybrid genes were transferred to infectious AcNPV expression vectors. Biologically active interferon was produced, and greater than 95% was secreted from infected insect cells. A maximum of ca. 5 X 10(6) U of interferon activity was produced by 10(6) infected cells. These results demonstrate that AcNPV should be suitable for use as a eucaryotic expression vector for the production of products from cloned genes.

Max D. Summers

Papers

The International Committee on Taxonomy of Viruses

Trends in the development of baculovirus expression vectors

Production of human beta interferon in insect cells infected with a baculovirus expression vector.

The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper

High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors