Susan E. Conrad

Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene.

We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, G0-phase cells, but increase dramatically (10- to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two- to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six- to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK- cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.

Sequence arrangement of tRNA genes on a fragment of drosophila melanogaster DNA cloned in E. coli

A plasmid with the vector Col E1 attached to an insert of Drosophila melanogaster DNA carrying four tRNA genes has been cloned in E. coli. Some features of the sequence arrangement and the positions of the tRNA genes have been determined by electron microscopic methods and by restriction endonuclease mapping. tRNA genes were mapped at 1.4, 4.7, 5.9 and 8.6 kb from one of the Drosophila/Col E1 junctions in the Drosophila insert of total length 9.34 kb. There are several secondary structure features consisting of inverted repeat sequences of length about 70-100 nucleotide pairs, some with and some without intervening loops, irregularly distributed on the insert. Cross-hybridization of tRNAs isolated by hybridization to separated restriction fragments indicate that the tRNA genes at 4.7, 5.9 and 8.6 kb are identical and differ from the one at 1.4 kb. Thus the positions of the genes, of the secondary structure features and of the restriction endonuclease sites all indicate that the spacers between the genes are not identical tandem repeats. In situ hybridization with cRNA transcribed from the plasmid showed localization at region 42A of chromosome 2R.

Induction of cellular thymidine kinase occurs at the mRNA level.

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.

Role of plasmid-coded RNA and ribonuclease III in plasmid DNA replication

An in vitro replication system has been used to study the control of DNA replication of the relaxed plasmids Col E1 and RSF1030. An RNA transcript approximately 100 nucleotides long is synthesized during the in vitro DNA replication reaction. This RNA is synthesized approximately 450 bp away from the origin of replication. A small insertion in the coding sequence for the RNA made from Col E1 DNA leads to a larger RNA species and simultaneously to an increase in plasmid copy number. Revertants missing the specific insertion show shorter RNA transcripts and wild-type copy number. Although plasmids Col E1 and RSF1030 have no extensive sequence homology, the RNA synthesized during RSF1030 replication has almost the same mobility as the Col E1 RNA on polyacrylamide gels and hybridizes to the Col E1 origin region. Extracts prepared from mutants of Escherichia coli deficient in ribonuclease III do not replicate RSF1030 or Col E1 plasmids in vitro. When supplemented with homogeneous RNAase III, such extracts do support DNA replication on these templates, indicating that RNAase III is required for DNA replication. We propose that the 100 nucleotide RNA species is involved in regulating the initiation of DNA replication of these plasmids, and that RNAase III may be involved in processing this RNA.

Estrogen increases brain expression of the mRNA encoding transthyretin, an amyloid β scavenger protein

Estrogen replacement therapy in postmenopausal women is associated with a reduced risk of Alzheimers Disease (AD). The multiple mechanisms by which estrogen protects against AD are still unknown. To conduct a broad screen for estrogen-regulated AD-related genes in the brain, we used cDNA array assays of brain mRNA samples from ovariectomized (ovx) adult female mice treated with either 17beta-estradiol or vehicle at 1 or 5 weeks post-ovx. The gene encoding transthyretin (TTR), which has been reported to scavenge amyloid beta peptides and reduce amyloid plaque formation, is increased by estradiol treatment at both 1 and 5 weeks post-ovx. Northern blot analyses and RNase protection assays performed on whole brain samples obtained from estradiol- or vehicle-treated mice confirmed the cDNA array assays showing a significant increase in TTR mRNA with estradiol treatment. Qualitative in situ hybridization or immunocytochemistry performed on brain sections demonstrated that TTR mRNA is expressed only in choroid plexus and leptomeninges, and that both estrogen receptor proteins, alpha and beta, are present in choroid plexus cells. These novel findings suggest that estrogen may reduce the risk of AD by acting on choroid plexus cells to increase TTR gene expression, leading to enhanced sequestration and reduced aggregation of amyloid beta peptides.

The sequence arrangement of Drosophila melanogaster 5s DNA cloned in recombinant plasmids.

Abstract Segments of Drosophila melanogaster DNA containing 5S rRNA genes have been propagated in recombinant plasmids using E. coli as a host and Col E1 as a vector. Electron microscope partial denaturation mapping, mapping by ferritin labeling and restriction enzyme-gel electrophoresis analysis all indicate that the Drosophila DNA inserts of these plasmids consist of tandem repeats of 5S genes and spacer regions. The repeat length is approximately 380 nucleotide pairs (ntp), corresponding to a gene of length 120 ntp and a spacer of length 260 ntp. The insert in one plasmid (pCIT9) consists of 32 contiguous repeats. Restriction enzyme-gel electrophoresis analysis shows that all these repeats have the same length within ± 5 nucleotides. This repeat length is estimated as 370 ± 20 ntp by gel electrophoresis and 390 ± 40 ntp by partial denaturation mapping. A second plasmid (pCIT19) contains three complete genes, two complete spacers and incomplete flanking spacer sequences. The two complete repeat units released by suitable restriction endonuclease digestions differ in length by 20 ± 5 ntp, with estimated lengths of 370 and 390 ntp. The positions and spacings of the genes on this plasmid have been observed directly by ferritin labeling and by partial denaturation mapping. The A+T content of the 5S DNA spacer region is calculated to be 68%. By in situ hybridization, cRNA transcribed from one plasmid hybridizes to polytene chromosomes only at band 56F, the known locus of the 5S rRNA genes. Spontaneous excision of some of the tandem repeat units from the recombinant plasmids occurs during growth in E. coli; the frequency of excision does not depend upon the recA character of the host, but is greatly increased by chloramphenicol treatment.

Identification of a G1-S-phase-regulated region in the human thymidine kinase gene promoter.

We have identified a regulatory region in the human thymidine kinase gene promoter. A set of promoter deletion mutants was constructed, linked to the bacterial neomycin resistance gene, and stably transfected into Rat3 cells. It was shown that the region between 135 and 67 base pairs upstream of the cap site is required for conveying G1-S-phase regulation to the linked neo gene. In addition, primer extension assays demonstrated that the same transcriptional start sites were used in G1- and S-phase cells and in the various deletion mutants tested.

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is an antiestrogen-regulated inhibitor of Cdk4 in human breast cancer cells

The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Antiestrogen treatment of MCF-7 cells causes dramatic decreases in both Cdk4 and Cdk2 activities, which leads to a G1phase cell cycle arrest. In this report, we investigate the mechanism(s) by which Cdk4 activity is regulated in MCF-7 cells. Through time course analysis, we demonstrate that changes in Cdk4 activity in response to estrogen or antiestrogen treatment do not correlate directly with cyclin D1 protein levels or association. In contrast, Cdk4 activity does correlate with changes in the level of the Cdk inhibitor p21WAF1/Cip1. Furthermore, we show that extracts of antiestrogen-treated cells contain a factor capable of inhibiting the Cdk4 activity present in extracts of estrogen-treated cells, and immunodepletion experiments identify this factor as p21WAF1/Cip1. These results identify p21WAF1/Cip1 as an important physiological regulator of Cdk4 complexes in human breast cancer cells.

Signal Transducer and Activator of Transcription 5a Mediates Mammary Ductal Branching and Proliferation in the Nulliparous Mouse

Signal transducer and activator of transcription (Stat)5a is a critical regulator of mammary gland development. Previous studies have focused on Stat5as role in the late pregnant and lactating gland, and although active Stat5a is detectable in mammary epithelial cells in virgin mice, little is known about its role during early mammary gland development. In this report, we compare mammary gland morphology in pubertal and adult nulliparous wild-type and Stat5a-/- mice. The Stat5a-null mammary glands exhibited defects in secondary and side branching, providing evidence that Stat5a regulates these processes. In addition, Stat5a-/- mammary glands displayed an attenuated proliferative response to pregnancy levels of estrogen plus progesterone (E+P), suggesting that it plays an important role in early pregnancy. Finally, we examined one potential mediator of Stat5as effects, receptor activator of nuclear factor-kappaB ligand (RANKL). Stat5a-/- mammary glands were defective in inducing RANKL in response to E+P treatment. In addition, regulation of several reported RANKL targets, including inhibitor of DNA binding 2 (Id2), cyclin D1, and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), was altered in Stat5a-/- mammary cells, suggesting that one or more of these proteins mediate the effects of Stat5a in E+P-treated mammary epithelial cells.

Progesterone receptor A-regulated gene expression in mammary organoid cultures.

Progesterone, through the progesterone receptor (PR), promotes development of the normal mammary gland and is implicated in the etiology of breast cancer. We identified PRA-regulated genes by microarray analysis of cultured epithelial organoids derived from pubertal and adult mouse mammary glands, developmental stages with differing progesterone responsiveness. Microarray analysis showed significant progestin (R5020)-regulation of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. Greater induction of receptor activator of NFkappaB ligand and calcitonin expression was observed in adult organoids, suggesting possible roles in the differential progesterone responsiveness of the adult and pubertal mammary glands. Analysis of the R5020-responsive transcriptome revealed several enriched biological processes including cell adhesion, immune response, and survival. R5020 both induced Agtr1 and potentiated angiotensin II-stimulated proliferation, highlighting the functional significance of the latter process. Striking up-regulation of genes involved in innate immunity processes included the leukocyte chemoattractants serum amyloid A1, 2 and 3 (Saa1, 2, 3). In vivo analysis revealed that progesterone treatment increased SAA1 protein expression and leukocyte density in mammary gland regions undergoing epithelial expansion. These studies reveal novel targets of PRA in mammary epithelial cells and novel linkages of progesterone action during mammary gland development.