Analyses of steroid receptors are important for understanding molecular details of transcriptional control, as well as providing insight as to how an individual transacting factor contributes to cell identity and function. These studies have led to the identification of a superfamily of regulatory proteins that include receptors for thyroid hormone and the vertebrate morphogen retinoic acid. Although animals employ complex and often distinct ways to control their physiology and development, the discovery of receptor-related molecules in a wide range of species suggests that mechanisms underlying morphogenesis and homeostasis may be more ubiquitous than previously expected.

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The steroid and thyroid hormone receptor superfamily

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.

High-efficiency transformation of mammalian cells by plasmid DNA.

https://limlab.ucsf.edu/papers/pdfs/lag_2013.pdf

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

Identification of complementary DNAs encoding the human glucocorticoid receptor predicts two protein forms, of 777 (alpha) and 742 (beta) amino acids, which differ at their carboxy termini. The proteins contain a cysteine/lysine/arginine-rich region which may define the DNA-binding domain. Pure radiolabelled glucocorticoid receptor, synthesized in vitro, is immunoreactive and possesses intrinsic steroid-binding activity characteristic of the native glucocorticoid receptor.

/pdf/primary-structure-and-expression-of-a-functional-human-9s709u9sel.pdf

Primary structure and expression of a functional human glucocorticoid receptor cDNA

To investigate the sequences necessary for proper initiation of transcription of SV40 early genes, we have constructed several deletion mutants in the promoter region. The TATA box region is apparently involved in fixing initiation precisely within a narrow area, but is dispensable for gene expression, while the sequences located more than 150 base pairs upstream are indispensable.

In vivo sequence requirements of the SV40 early promoter region

Regions of allelic hypervariability in the murine Aα immune response gene

The murine Eα immune response gene

The present invention provides methods for inhibiting pathways induced by commensal bacteria of the gastrointestinal (GI) tract that lead to Th 17 differentiation, which in turn leads to localized and systemic accumulation of Thl7 cells that are causally associated with inflammatory and autoimmune disorders and methods for identifying agents useful for treating non-gut autoimmune disorders.

Methods for modulating autoimmunity

Methods of diagnosing, screening and treating a subject having an autoimmune disorder include evaluating of modulating the alternative pathway of complement activation.

Christophe Benoist

Papers

In vivo sequence requirements of the SV40 early promoter region

Regions of allelic hypervariability in the murine Aα immune response gene

The murine Eα immune response gene

Methods for modulating autoimmunity

Methods of treating and diagnosing arthritis