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.

Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.

https://www.pnas.org/content/pnas/89/12/5547.full.pdf

Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

Mutations in the p53 tumour-suppressor gene are the most frequently observed genetic lesions in human cancers. To investigate the role of the p53 gene in mammalian development and tumorigenesis, a null mutation was introduced into the gene by homologous recombination in murine embryonic stem cells. Mice homozygous for the null allele appear normal but are prone to the spontaneous development of a variety of neoplasms by 6 months of age. These observations indicate that a normal p53 gene is dispensable for embryonic development, that its absence predisposes the animal to neoplastic disease, and that an oncogenic mutant form of p53 is not obligatory for the genesis of many types of tumours.

Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours

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.

Human T-lymphotropic virus type 1 (HTLV-1) is a suspected causative agent of adult T-cell leukemia. One of the viral genes encodes a protein (tat) that not only results in transactivation of viral gene expression but may also regulate the expression of certain cellular genes that are important for cell growth. Transgenic mice that expressed the authentic tat protein under the control of the HTLV-1 long terminal repeat were generated, and cell types that are permissive for the viral promoter and the effects of the tat gene on these cells were studied. Three of eight founder mice with high levels of expression of the transgene in muscle were bred and then analyzed. All developed soft tissue tumors at multiple sites between 13 to 17 weeks of age. This phenotype was transmitted to nine of nine offspring that inherited the tat gene and were available for analysis. The remaining five founders expressed the transgene in the thymus, as well as in muscle. This second group of mice all exhibited extensive thymic depletion and growth retardation; in all of these mice, death occurred between 3 to 6 weeks of age before tumors became macroscopically visible. The tat gene under the control of the HTLV-1 regulatory region showed tissue-specific expression and the tat protein efficiently induced mesenchymal tumors. The data establish tat as an oncogenic protein and HTLV-1 as a transforming virus.

https://science.sciencemag.org/content/sci/237/4820/1324.full.pdf

The tat Gene of Human T-Lymphotropic Virus Type 1 Induces Mesenchymal Tumors in Transgenic Mice

The simian virus (SV40) 72-base pair (bp) tandem repeated sequences have recently been shown to function as activators or enhancers of early viral transcription. A recombinant viral genome was recently constructed by inserting 72-bp tandem repeats from the Moloney murine sarcoma virus (MSV) in place of the 72-bp repeats of SV40. Although this genome replicates in monkey kidney cells, its rate of large tumor antigen expression and replication is considerably slower than that of wild-type SV40. In mouse cells, however, equivalent levels of large tumor antigen appear to be expressed from both wild-type and recombinant genomes, suggesting a relationship between the level of enhancer activity and the host cell. To confirm this observation, we have applied a sensitive quantitative assay for gene expression based on the conversion of chloramphenicol to its acetylated forms. The gene encoding the enzymatic function chloramphenicol acetyltransferase was inserted into two vectors in which the enhancer sequences from SV40 or MSV were placed adjacent to the early SV40 promoter. The SV40 tandem repeats appear to activate gene expression to significantly higher levels in monkey kidney cells, but the MSV repeats are more active in two lines of mouse cells. These findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.

Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus.

On the late side of the simian virus 40 (SV40) DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base pairs long. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from which one of the 72-base-pair repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability, as recognized by the absence of early transcription and of tumor antigen production. These observations indicate that the 72-base-pair repeated sequences form an essential element in the early viral transcriptional promoter and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA. In a parallel experiment it was found that the extended deletion mutant was also unable to complement a late temperature-sensitive mutant of SV40, tsB. This suggests that the extended mutant is also defective in DNA replication or late transcription (or both).

https://www.pnas.org/content/pnas/78/2/943.full.pdf

Simian virus 40 tandem repeated sequences as an element of the early promoter

The classical transplantation antigens (the major histocompatibility complex class I antigens) play a key role in host defense against cells expressing foreign antigens. Several naturally occurring tumors and virally transformed cells show an overall suppression of these surface antigens. Since the class I molecules are required in the presentation of neoantigens on tumor cells to the cytotoxic T lymphocytes, their absence from the cell surface may lead to the escape of these tumors from immunosurveillance. To test this possibility, a functional class I gene was transfected into human adenovirus 12-transformed mouse cells that do not express detectable levels of class I antigens; the transformants were tested for expression of the transfected gene and for changes in oncogenicity. The expression of a single class I gene, introduced by DNA-mediated gene transfer into highly tumorigenic adenovirus 12-transformed cells, was sufficient to abrogate the oncogenicity of these cells. This finding has important implications for the regulation of the malignant phenotype in certain tumors and for the potential modulation of oncogenicity through derepression of the endogenous class I genes.

https://science.sciencemag.org/content/sci/228/4695/26.full.pdf

Reversal of oncogenesis by the expression of a major histocompatibility complex class I gene

To investigate whether DNA viruses can augment gene expression of the human immunodeficiency virus (HIV), cotransfection experiments were carried out in which a recombinant plasmid containing the HIV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene was transfected into cultured cells along with plasmids containing DNA from various distinct classes of DNA viruses. Molecular clones containing JC virus, BK virus, lymphotropic papovavirus, bovine papilloma virus, type 1 herpes simplex virus (HSV-1), and varicella-zoster virus sequences increased CAT expression directed by the HIV LTR. Trans-activation of the HIV LTR varied in different cell lines, but in each case the HIV tat gene product elicited the greatest stimulation. Primer-extension assays specific for HIV LTR mRNA revealed increased levels of steady-state RNA following transfection with HIV tat as well as with several of the DNA viruses. Virus-specific RNA expression paralleled the stimulation of CAT activity. More-than-additive effects were observed at both the RNA and protein levels when tat plus type 1 herpes simplex virus DNAs or tat plus JC virus DNAs were transfected into cells with the HIV LTR-CAT plasmid. These data suggest that coinfection of cells by HIV and some DNA viruses can stimulate the expression of HIV.

George Khoury

Papers

The tat Gene of Human T-Lymphotropic Virus Type 1 Induces Mesenchymal Tumors in Transgenic Mice

Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus.

Simian virus 40 tandem repeated sequences as an element of the early promoter

Reversal of oncogenesis by the expression of a major histocompatibility complex class I gene

Trans-activation of the human immunodeficiency virus long terminal repeat sequence by DNA viruses.