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Biomedical subjects

S R Ross

Publications and source records attributed to S R Ross.

At least 19 recordsLinked to original sources
PubMed1992

Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.

Transgenic mice were produced containing the adipocyte-specific regulatory region from the adipocyte P2 (aP2) gene linked to the simian virus 40 transforming genes. Most of the transgenic mice developed brown fat tumors (hibernomas) in their interscapular brown adipose tissue. Hibernoma formation was noticeable in some of the mice as early as 1 day after birth and most of the mice developed very large tumors by 1 month of age. All of the tumor tissue expressed the brown fat-specific uncoupling protein (UCP) gene as well as the aP2 gene. Several of the tumors have been used to establish cultured cell lines and at least one of these lines can be induced to differentiate into brown adipocytes. The cultured adipocytes express mRNA for UCP upon stimulation with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, norepinephrine, isoproterenol or D7114, a beta 3 adrenergic agonist. Thus, regulation of the key thermogenic gene UCP can now be studied in an established cell line.

Adipose Tissue, Brown↗
PubMed1992

Transgenic mouse mammary tumor virus superantigen expression prevents viral infection.

Endogenous mouse mammary tumor virus (MMTV) proviruses have recently been shown to cosegregate genetically with the minor lymphocyte-stimulating loci, also termed self-superantigens. The antigenic activity has been localized to the open reading frame (ORF) protein encoded in the long terminal repeat of MMTV. We show here that unlike their nontransgenic littermates, transgenic mice expressing high levels of an ORF protein derived from the C3H exogenous MMTV specifically delete their V beta 14+ T cells and do not become infected with this virus when it is present in their mother's milk. Thus, it appears that MMTV utilizes cells of the immune system in its infection pathway, and mice that retain endogenous MMTVs should be immune to infection by exogenous virus. These results offer possible new approaches to anti-viral therapy or immunization.

Animals↗
PubMed1992

Placental-specific expression from the mouse placental lactogen II gene promoter.

The gene for mouse placental lactogen II (mPL-II) has been isolated and characterized. This gene contains five exons, with a transcription start site 59 nucleotides upstream of the translation initiation ATG. Introduction of a DNA construct containing 2.7 kilobases of sequence upstream of the mPL-II transcription initiation site directed the synthesis of a linked coding region for the simian virus 40 large and small tumor antigens in placental trophoblast giant cells of transgenic mice. The pattern of simian virus 40 transgene expression in the placenta was indistinguishable from that of the endogenous mPL-II gene. In contrast, the first 569 base pairs upstream of the transcription start site proved insufficient to direct placental expression. Thus, one or more elements required for placental trophoblast giant cell expression have been localized to a region between -2700 and -569 of the mPL-II gene.

Amino Acid Sequence↗
PubMed1992

Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells.

Inoculated immunogenic cancer cells after initial growth are potentially rejected by specific host immunity; however, the outcome of the interaction between host and inoculated cancer cells is a function of multiple factors including the route of inoculation, the number of cells, the density of antigens on the injected cancer cells, and the state of the immune system of the host. In the present study, we have examined a different kind of variable: the stroma that inoculated tumor cells initially reside in. The impetus to examine this factor arises from observations that cancer cells from several lines inoculated as fragments of solid tumors often grow progressively, whereas the same number or more than 10-fold larger numbers of identical type cells injected as a suspension are rejected, even though fragments or suspended cells are both tumorigenic at the same doses in nude mice. In the present studies, we found that: (a) indeed, cancer cells inoculated as fragments were more tumorigenic than cancer cells in suspension; (b) the tumorigenicity of suspended cancer cells was increased by injection of the cells into polyurethane sponge implants; (c) cancer cells were more tumorigenic embedded in syngeneic stroma than in transgenic antigenic stroma expressing the K216 major histocompatibility complex class I antigen; and (d) antigenic, bone marrow-derived, stromal components (presumably passenger leukocytes) were sufficient to cause rejection of immunogenic but antigenically unrelated cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Identification of a fat cell enhancer: analysis of requirements for adipose tissue-specific gene expression.

The molecular basis for adipose-specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5'-flanking region of the adipocyte P2 (aP2) gene to direct cell-type specific gene expression. Although the proximal promoter containing AP-1 and C/EBP binding sites is capable of directing differentiation-dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that -5.4 kb of the 5'-flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at -5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation-dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis- and trans- acting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.

Adipose Tissue↗
PubMed1992

A mouse mammary tumor virus mammary gland enhancer confers tissue-specific but not lactation-dependent expression in transgenic mice.

The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) is known to contain a number of transcriptional regulatory elements, including glucocorticoid response elements. In this study, we showed that a mammary gland/salivary gland enhancer found in the LTR of this virus directs expression of a heterologous promoter to both virgin and lactating mammary glands in transgenic mice. Using transgenic mice containing hybrid gene constructs with various deletions of the LTR sequences linked to marker genes, we also showed that the dramatic increase in MMTV expression that occurs during lactation is due to the glucocorticoid response elements. Thus, the MMTV LTR encodes two distinct elements, both of which are required for a high level of expression in lactating mammary glands.

Animals↗
PubMed1991

Identification of a potent adipocyte-specific enhancer: involvement of an NF-1-like factor.

The molecular basis for adipocyte-specific gene expression is not known. We have demonstrated that while short (-168) segments of the 5'-flanking sequence of the adipocyte P2 gene containing AP-1- and C/EBP-binding sites can direct expression of a heterologous gene in cultured adipocytes, they cannot support tissue-specific expression in a transgenic mouse. We have therefore analyzed larger segments of the aP2 5'-flanking region by transfection into adipocytes and have found an enhancer at -5.4 kb. This 500-bp enhancer directs expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in a differentiation-dependent fashion when linked to its own minimal promoter or to an enhancerless SV40 promoter. Moreover, this enhancer stimulates very strong and highly specific expression from the CAT gene in the adipose tissues of transgenic mice. A smaller fragment (190 bp) having enhancer activity in adipocytes was defined and demonstrated to contain a binding site for an abundant nuclear protein. This factor has the binding specificity and several other properties characteristic of the nuclear factor 1 (NF-1) transcription/replication factor family, and mutation of this NF-1-binding site greatly reduces the function of the 500-bp enhancer. These results identify and characterize the first functional enhancer with specificity for adipose cells and also demonstrate that a member(s) of the NF-1 family is involved in adipocyte-specific gene expression.

Adipose Tissue↗
PubMed1990

Animals bearing malignant grafts reject normal grafts that express through gene transfer the same antigen.

Breaking the state of immunological unresponsiveness of tumor-bearing individuals to cancer is a prerequisite for active or passive tumor-specific immunotherapy. To study this problem the immunogenic MHC class I antigen, K216 was transfected into a progressor tumor. The transfected tumors were regularly rejected by normal mice but grew progressively in mice bearing nontransfected tumors. In addition, transgenic mice were derived to obtain normal cells and tissues expressing the same K216 gene product. Normal mice rejected K216-positive normal or malignant tissue grafts and generated K216-specific CTL in vitro and in vivo in response to these challenges. In contrast, mice bearing nontransfected tumors, though rejecting K216-positive nonmalignant tissue grafts, did not reject K216-positive tumors nor generate K216-specific CTL in response to K216-positive tumor cells. Mice bearing K216-positive tumors also rejected the nonmalignant K216-positive tissue grafts, but this in vivo response failed to lead to rejection of the simultaneously present tumor graft expressing the same antigen; in fact, immunity had no measurable effect whatsoever on tumor size or incidence and caused no selection for antigen loss variants. Taken together, the present findings suggest that transfer of expression of a target antigen into nonmalignant cells provides a way for obtaining effective stimulation of antigen-specific CTL in tumor-bearing mice, but that additional manipulations will be required to cause immunological rejection of established tumors.

Animals↗
PubMed1990

GC-rich murine adenosine deaminase gene promoter supports diverse tissue-specific gene expression.

The murine adenosine deaminase (ADA) gene has a GC-rich promoter that is structurally typical of many mammalian "housekeeping" gene promoters. The ability of the ADA gene promoter to support diverse tissue-specific gene expression was investigated. Endogenous ADA gene expression in different mouse tissues was found to vary over a greater than 3000-fold range in a highly complex pattern. This range of expression was also observed in cultured human cell lines derived from different tissues. The ADA levels in all tissues and cell lines examined correlated closely with steady-state ADA mRNA levels. Several of the mouse tissues examined also showed stage-specific variation during postnatal development. In order to determine whether tissue-specific ADA expression was controlled by cis-acting sequences upstream of the coding region, constructs containing a reporter gene regulated by the ADA gene's 5' flanking sequences were used to generate transgenic mice. All transgene-expressing mice obtained showed diverse reporter gene expression in the tissues analyzed. Our results demonstrate that both in vivo and in the context of an integrated transgene this GC-rich promoter can support highly diverse gene expression in all tissues of the animal.

Adenosine Deaminase↗
PubMed1990

A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo.

The murine gene for adipocyte P2 encodes an adipocyte-specific member of the family of intracellular lipid binding proteins. The region upstream from the start of transcription of this gene has been found to contain binding sites for the transcription factors c-jun/c-fos and C/EBP (CCAAT/enhancer binding protein) and several short sequence elements found in other adipocyte gene promoters, termed fat-specific elements. To identify DNA sequences that were responsible for the high level of transcription of the gene for adipocyte P2 in vivo, we made a series of transgenic mice containing 168 base pairs (bp), 247 bp, 1.7 kilobases (kb), and 5.4 kb of 5' flanking sequence linked to the bacterial gene chloramphenicol acetyltransferase. Although plasmids containing only 168 bp of 5' sequence including the C/EBP and AP-1 (activation protein 1) binding sites were expressed well in cultured adipocytes, high levels of chloramphenicol acetyltransferase activity in the adipose tissue of transgenic mice were not observed until the 5' flanking region was extended to kb -54. An enhancer mapping between kb -4.9 and kb -5.4 upstream from the start of transcription was identified by transfection of further deletions into cultured adipocytes. This enhancer, when linked to a bp -63 promoter fragment from the gene for adipocyte P2, directed very high level chloramphenicol acetyltransferase expression specifically to adipose tissue in transgenic mice. These results identify a functional adipose-specific enhancer and indicate that it is the major determinant of tissue specificity of the gene for adipocyte P2. These results also demonstrate that the proximal-promoter binding sites for AP-1 and C/EBP are not sufficient or necessary to give adipose-tissue-specific expression in vivo, though they may play an important role in the response of this promoter to glucocorticoids.

Adipose Tissue↗
PubMed1990

Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice.

Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene.

Animals↗
PubMed1990

Craniofacial dysmorphogenesis in transgenic mice.

While using transgenic mice to study the regulation of alpha-fetoprotein (AFP) it was noted that two different alpha-fetoprotein-chloramphenicol acetyl transferase (CAT) transgenes resulted in the appearance of craniofacial anomalies in 11% of the offspring derived from crosses between transgenic mice and nontransgenic mates. A total of 13 fetuses exhibited abnormalities; two are described in detail. Ninety-two percent of the affected fetuses had some form of mandibular abnormality while zygomatic and ossicular defects appeared in more than 40% of the specimens. Aglossia and aberrant musculature were also present in the most severely involved specimen. Eight of the affected fetuses were screened for the presence of the AFP-CAT plasmid and all were found to be heterozygous for the transgene. Since the probability that all 8 of the abnormal fetuses known to carry the CAT gene would have done so by chance was only 1 in 256, it may be assumed that these anomalies did not appear spontaneously, but were somehow created by the transgenic procedure. It is not known how the transgenic material led to the observed dysmorphogenetic pattern, but theoretically introduction of the AFP-CAT plasmid could have disrupted morphogenesis through the presence of the "foreign" CAT protein or a decrease in the availability of AFP. Since AFP levels were found to be normal in both the liver and the yolk sac of transgenic fetuses, it appears that the presence of CAT was responsible for the craniofacial anomalies described here.

Animals↗
PubMed1989

Obesity-linked regulation of the adipsin gene promoter in transgenic mice.

The mouse adipsin gene encodes a member of the serine protease family that is expressed predominantly in adipose tissue and is secreted into the bloodstream. Adipsin expression is sharply down-regulated in several models of genetic and acquired obesity, representing the first example of an adipocyte gene whose expression is greatly altered in this disorder. In this study, we have asked whether a DNA fragment from the adipsin gene can direct tissue-specific expression of a heterologous gene and mediate the suppression of this expression in genetic and chemically induced obesity. Transgenic mice have been constructed with 950 bases of DNA from the 5' flanking region of the adipsin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene in a mouse strain bearing a recessive obesity gene (diabetes, db). By crossing db/+ transgenic mice with nontransgenic db/+ mice, we obtained progeny that allowed a direct comparison of CAT expression in the tissues of lean and obese littermates. The lean mice express CAT activity predominantly in adipose tissue, while the obese mice show a marked reduction in CAT expression relative to the lean controls. When similar experiments are performed with an adipsin-CAT fusion gene containing a heterologous AKV (AKR mouse leukemia virus) enhancer, the tissue specificity of CAT expression in lean mice is broadened to include the thymus, spleen, brain, and other tissues; down-regulation occurs in all of these tissues in mice homozygous for the obesity gene or in mice that have been injected with monosodium glutamate (MSG), which induces obesity. These results indicate that 950 bases of the 5' flanking region of the adipsin gene carry information that specifies both expression in adipose tissue and a response to a gene or chemical that induces obesity. These results also suggest that the trans-acting factors that are regulated aberrantly in these forms of obesity are not restricted to adipose tissue and could play a role in obesity-linked dysfunctions observed in other tissues as well.

Animals↗
PubMed1988

Endogenous mouse mammary tumor virus is expressed in several organs in addition to the lactating mammary gland.

We studied the transcription of endogenous mouse mammary tumor virus (MMTV) in transgenic and normal strains of mice of different genetic backgrounds. Although the lactating mammary gland in all strains showed the highest level of MMTV expression, detectable levels of viral RNA were also found in the lungs, kidneys, salivary glands, seminal vesicles and/or prostate gland, testes, and lymphoid tissue in mice which contain different endogenous proviruses. Transcription in transgenic mice containing the MMTV long terminal repeat linked to the simian virus 40 early region also occurred in these same organs. These results indicate that the MMTV long terminal repeat functions in several organs in addition to the lactating mammary gland.

Animals↗
PubMed1988

Requirement for the simian virus 40 small tumor antigen in tumorigenesis in transgenic mice.

To examine the role of simian virus 40 (SV40) large T and small t antigens in tumorigenesis in animals, we generated transgenic mice which expressed either both the SV40 large T and small t antigens or the SV40 large T antigen alone under the control of the mouse mammary tumor virus long terminal repeat. The mouse mammary tumor virus long terminal repeat directs the expression of transgenes in ductal epithelial cells of several organs, including the mammary gland, lung, and kidney, and in lymphoid cells. The mice which expressed both the T and t tumor antigens developed lung and kidney adenocarcinomas, while those which expressed large T alone did not. Both types of mice developed malignant lymphomas with similar frequencies and latency periods. Our results show that the SV40 small t antigen cooperates with the large T antigen in inducing tumors in slowly dividing epithelial cells in the lung and kidney.

Animals↗
PubMed1987

The mouse mammary tumor virus long terminal repeat directs expression in epithelial and lymphoid cells of different tissues in transgenic mice.

A series of transgenic mice was developed that contained the simian virus 40 early region genes under the transcriptional control of the mouse mammary tumor virus long terminal repeat, including the promoter and glucocorticoid response elements. These mice all expressed the transgene in the epithelial cells of a number of different organs, such as lungs, kidneys, and prostate, salivary, and mammary glands, and in Leydig and lymphoid cells. Transcription of the chimeric gene was inducible by glucocorticoids, either after transfection into tissue culture cells or in cells cultured from animals carrying the transgene. Many, but not all, tissues which expressed the simian virus 40 sequences, as determined immunologically and by RNA analysis, developed into tumors, although they showed premalignant features. Since the mouse mammary tumor virus long terminal repeat is expressed in a number of different cell types when inherited through the germ line, the lactating mammary gland-specific transcription of endogenous proviruses must require other factors or sequences to achieve this specificity.

Animals↗