Mouse Hprt Research Articles

Genotoxicity testing of nanomaterials.

Nanomaterials have outstanding and unprecedented advantageous material properties but may also cause adverse effects in humans upon exposure. Testing nanomaterials for genotoxic properties is challenging because traditional testing methods were designed for small, soluble molecules and may not be easily applicable without modifications. This review critically examines available genotoxicity tests for use with nanomaterials, including DNA damage tests such as the comet assay, gene mutation tests such as the mouse lymphoma and hprt assay, and chromosome mutation tests such as the micronucleus test and the chromosome aberration test. It presents arguments for the relative usefulness of various tests, such as preferring the micronucleus test over the chromosome aberration test for scoring chromosome mutations and preferring mammalian cell gene mutation tests because the Ames test has limited utility. Finally, it points out the open questions and further needs in adapting genotoxicity tests for nanomaterials, such as validation, reference nanomaterials, and the selection of top test concentrations, as well as the relevance and applicability of test systems and the need to define testing strategies. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Silicon-Nanotube-Mediated Intracellular Delivery Enables Ex Vivo Gene Editing.

Engineered nano-bio cellular interfaces driven by vertical nanostructured materials are set to spur transformative progress in modulating cellular processes and interrogations. In particular, the intracellular delivery-a core concept in fundamental and translational biomedical research-holds great promise for developing novel cell therapies based on gene modification. This study demonstrates the development of a mechanotransfection platform comprising vertically aligned silicon nanotube (VA-SiNT) arrays for ex vivo gene editing. The internal hollow structure of SiNTs allows effective loading of various biomolecule cargoes; and SiNTs mediate delivery of those cargoes into GPE86 mouse embryonic fibroblasts without compromising their viability. Focused ion beam scanning electron microscopy (FIB-SEM) and confocal microscopy results demonstrate localized membrane invaginations and accumulation of caveolin-1 at the cell-NT interface, suggesting the presence of endocytic pits. Small-molecule inhibition of endocytosis suggests that active endocytic process plays a role in the intracellular delivery of cargo from SiNTs. SiNT-mediated siRNA intracellular delivery shows the capacity to reduce expression levels of F-actin binding protein (Triobp) and alter the cellular morphology of GPE86. Finally, the successful delivery of Cas9 ribonucleoprotein (RNP) to specifically target mouse Hprt gene is achieved. This NT-enhanced molecular delivery platform has strong potential to support gene editing technologies.

Variable transcriptional regulation of the human aldosterone synthase gene causes salt-dependent high blood pressure in transgenic mice.

Aldosterone, synthesized in the adrenal cortex by the enzyme CYP11B2, induces positive sodium balance and predisposes to hypertension. Various investigators, using genomic DNA analyses, have linked -344T polymorphism in the human CYP11B2 (hCYP11B2) gene to human hypertension. hCYP11B2 gene promoter has 3 single-nucleotide polymorphisms in linkage disequilibrium: T/A at -663, T/C at -470, and C/T at -344. Variants ACT occur together and form the haplotype-I (Hap-I), whereas variants TTC constitute Hap-II. We hypothesize that these single-nucleotide polymorphisms, when present together, will lead to haplotype-dependent differences in the transcriptional regulation of the hCYP11B2 gene and affect blood pressure regulation. We evaluated differences in tissue expression in vivo and consequential effects on blood pressure stemming from the 2 haplotypes. Novel transgenic mice with the hCYP11B2 gene, targeted to the mouse HPRT locus, with either Hap-II or Hap-I variant are used in this study. Our results show increased adrenal and renal expression of hCYP11B2 in transgenic mice with Hap-I when compared with mice with Hap-II. Importantly, we observed increased baseline blood pressure in Hap-I transgenic mice, an effect accentuated by a high-salt diet. Pathophysiological effects of elevated aldosterone were corroborated by our results showing upregulation of proinflammatory markers in renal tissues from the transgenic mice with Hap-I. These findings characterize the haplotype-dependent regulation of the hCYP11B2 gene where -344T serves as a reporter polymorphism and show that Hap-I leads to increased expression of hCYP11B2, with permissive effects on blood pressure and inflammatory milieu.

Tissue selective expression of conditionally‐regulated ROCK by gene targeting to a defined locus

ROCK kinases regulate actin-myosin structures downstream of Rho GTPases. We generated mice expressing 4-hydroxytamoxifen (4HT)-regulated human ROCK II (ROCKII:mER) under the transcriptional control of the cytokeratin14 (K14) promoter. The K14-ROCKII:mER minigene was recombineered into a novel cloning vector containing the promoter and first exon of the human HPRT gene, and second and third exons of the mouse Hprt gene. Homologous recombination into the Hprt locus, which is deleted for the promoter and first two exons in HM1 embryonic stem cells, reconstitutes a functional Hprt gene, allowing for growth in HAT (hypoxanthine-aminopterin-thymidine) medium. K14-promoter-driven ROCKII:mER expression was restricted to a superficial cell layer in embryoid bodies, with increased ROCK substrate phosphorylation induced by 4HT. ROCKII:mER-expressing primary murine keratinocytes responded to 4HT with increased substrate phosphorylation and cytoskeleton rearrangements, indicating that ROCKII:mER activity is regulated by 4HT in the target tissue. K14-ROCKII:mER mice will be valuable for examining the role of ROCK in skin development and cancer.

Two Major Factors Involved in the Reverse Dose-rate Effect for Somatic Mutation Induction are the Cell Cycle Position and LET Value

To study mechanisms which could be involved in the reverse dose rate effect observed during mutation induction after exposure to high LET radiation, synchronized mouse L5178Y cells were exposed to carbon 290 MeV/n beams with different LET values at the G2/M, G1, G1/S or S phases in the cell cycle. The frequency of Hprt-deficient (6-thioguanine-resistant) mutant induction was subsequently determined. The results showed that after exposure to high LET value radiation (50.8 and 76.5 keV/microm), maximum mutation frequencies were seen at the G2/M phase, but after exposure to lower LET radiation (13.3 keV/microm), the highest mutation frequencies were observed at the G1 phase. The higher LET beam always produced higher mutation frequencies in the G2/M phase than in the G1 phase, regardless of radiation dose. These results suggest that cells in the G2/M phase is hyper-sensitive for mutation induction from high LET radiation, but not to mutation induction from low LET radiation. Molecular analysis of mutation spectra showed that large deletions (which could include almost entire exons) of the mouse Hprt gene were most efficiently induced in G2/M cells irradiated with high LET radiation. These entire exon deletions were not as frequent in cells exposed to lower LET radiation. This suggests that inappropriate recombination repair might have occurred in response to condensed damage in condensed chromatin in the G2/M phase. In addition, by using a hyper-sensitive mutation detection system (GM06318-10 cells), a reverse dose-rate effect was clearly observed after exposure to carbon beams with higher LET values (66 keV/microm), but not after exposure to beams with lower LET values (13.3 keV/microm). Thus, G2/M sensitivity towards mutation induction, and the dependence on radiation LET values could both be major factors involved in the reverse dose rate effect produced by high LET radiation.

Isx Participates in the Maintenance of Vitamin A Metabolism by Regulation of β-Carotene 15,15′-Monooxygenase (Bcmo1) Expression

Isx (intestine specific homeobox) is an intestine-specific transcription factor. To elucidate its physiological function, we generated Isx-deficient mice by knocking in the beta-galactosidase gene (LacZ) in the Isx locus (IsxLacZ/LacZ mice). LacZ staining of heterozygous (IsxLacZ/+) mice revealed that Isx was expressed abundantly in intestinal epithelial cells from duodenum to proximal colon. Quantitative mRNA expression profiling of duodenum and jejunum showed that beta-carotene 15,15'-monooxygenase (EC1.14.99.36 Bcmo1) and the class B type I scavenger receptor, which are involved in vitamin A synthesis and carotenoid uptake, respectively, were drastically increased in IsxLacZ/LacZ mice. Although mild vitamin A deficiency decreased Isx expression in duodenum of wild-type (Isx+/+) mice, severe vitamin A deficiency decreased Isx mRNA expression in both duodenum and jejunum of Isx+/+ mice. On the other hand, vitamin A deficiency increased Bcmo1 expression in both duodenum and jejunum of Isx+/+ mice. However, Bcmo1 expression was not increased in duodenum of IsxLacZ/LacZ mice by mild vitamin A deficiency. These data suggest that Isx participates in the maintenance of vitamin A metabolism by regulating Bcmo1 expression in the intestine.

23. Frequency of Random and Targeted Chromosomal Integration of Helper-Dependent Adenoviral Vector

For gene therapy of inherited diseases, integration of a therapeutic gene into random sites on chromosomes could potentially lead to serious problems such as cellular transformation and gene silencing. Homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to obtain safe and stable gene expression stably. We constructed helper-dependent adenoviral vectors (HD AdVs) and examined the ability of AdVs to correct an insertional mutation in exon 3 of the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse embryonic stem (ES) cells. These HD AdV contained the wild-type exon 3 sequence of the mouse Hprt gene with 6.7 kb (HD AdHprt6.7) or 18.6 kb (HD AdHprt18.6) of flanking intronic sequences, as well as the |[beta]|-geo marker gene. HR and random integration (RI) frequencies were calculated by dividing the total number of HAT- or neomycin-resistant colonies by the total number of cells plated. HR was only obtained by using the HD AdHprt18.6 vector, and the frequency was nearly 0.0002 per infected cell at an MOI of 1000. In the case of the HD AdHprt6.7 vector, no HR events were detected. The HR frequency by electroporation of a plasmid construct identical to the HD AdHprt18.6 vector was 23-fold less than that by vector infection. The structure of the exon 3 region of Hprt locus was analyzed in HAT-resistant ES clones by Southern analysis, and all the clones showed a pattern of faithful HR. Since the number of target cells might be limited in ex vivo gene therapy, such as stem cell therapy, we next investigated whether HD AdV-mediated HR can be efficiently achieved even in smaller cell populations. We successfully obtained HAT-resistant colonies at the frequency of 2.1 |[times]| 10|[minus]|4 per infected cell in a 24-well dish (25 HAT-resistant colonies out of 1.2 |[times]| 10|[minus]|5 cells infected, on average). These results clearly indicate gene correction can be achieved relatively efficiently even in smaller cell populations using AdVs. Both HD AdVs achieved similar RI frequencies of 10|[minus]|4 |[minus]| 10|[minus]|3 per cell, which was measured as a frequency to produce G418-resistant colonies. In contrast to the HR frequency, which showed an MOI-dependent increase, the RI frequency reached a plateau at an MOI of 100. In order to directly detect integrated vectors at DNA levels, we next performed PCR on ES clones infected with HD AdHprt18.6 and formed in the absence of G418 selection. Surprisingly, vector DNA was detected in 4 of the 74 samples (5 % of colonies). These results indicate that the actual RI frequency of HD AdVs is much higher than previously believed. Next, we determined the integration sites of the HD AdHprt18.6 vector by adaptor-ligated PCR. In contrast to retroviral, lentiviral and adeno-associated viral vectors, which tend to integrate near or within active genes, HD AdV had a tendency to integrate in intergenic regions (12 of 17 integrants, 71%). Small deletions of integrated vector DNA were found at the end of ITRs (16 of 17 integrations, 94%), ranging from 1 to 30 bp in size. These findings suggest that HR mediated by HD AdV is efficient and safe, and might be a viable option for ex vivo gene therapy of stem cells.

Pms2 deficiency results in increased mutation in the Hprt gene but not the Tk gene of Tk+/- transgenic mice

The effects of deficiency in the DNA mismatch repair (MMR) protein Pms2 were investigated using the endogenous mouse Hprt and Tk genes as reporters of intragenic mutation and loss of heterozygosity (LOH). Pms2(-/-)Tk(+/-), Pms2(+/+)Tk(+/-), Pms2(+/-)Tk(+/-) and Pms2(-/-)Tk(-/-) mice were bred from Pms2(+/-)Tk(+/-) mice. At 2 months of age, the body weight and splenic T lymphocyte yields were significantly lower in Pms2(-/-)Tk(-/-) mice than in littermates of the other genotypes. The mice were evaluated for their spontaneous mutant frequencies in the Hprt and Tk genes of splenic lymphocytes and their frequency of micronuclei in polychromatic erythrocytes from bone marrow. The cloning efficiency of lymphocytes derived from Pms2(-/-)Tk(-/-) animals was 12-fold lower than that of animals of the other genotypes. Compared with Pms2(+/+) and Pms2(+/-) mice, Pms2(-/-) mice had a 21- to 69-fold increase in the Hprt mutant frequency. The Hprt mutant frequency was equally high in Pms2-deficient, Tk(+/-) and Tk(-/-) mice. No significant Pms2-dependent change in mutant frequency was detected using the Tk mutational target. When individual Tk mutants were analyzed for LOH mutation by allele-specific genotyping, the fraction of LOH mutants was lower in Pms2-deficient than in Pms2-proficient mice (29.2 and 43.6%, respectively). The frequency of bone marrow micronuclei was significantly higher in Pms2(-/-)Tk(-/-) mice than in Pms2(-/-)Tk(+/-) mice. These observations suggest that the simultaneous occurrence of Tk and Pms2 deficiencies may cause a decrease in cell viability that diminishes the Tk mutational response, making it impossible to discern clearly the effect of Pms2 deficiency on LOH-type mutation using the Tk reporter system. The interaction between Tk deficiency and a component of the MMR system suggests that Tk-deficient cells may have higher levels of DNA polymerase misincorporation or endogenous DNA damage than Tk-proficient cells.

Nucleocytoplasmic transport signals affect the age at onset of abnormalities in knock-in mice expressing polyglutamine within an ectopic protein context.

In order to better understand the role of nuclear localization of polyglutamine in the human CAG repeat disorders, gene targeting was used to add either nuclear localization (NLS) or nuclear export (NES) signals to versions of the mouse Hprt protein containing expanded polyglutamine (HprtQ150). The NLS increased levels of nuclear HprtQ150 protein in the mouse brain and hastened both the presentation of neuronal intranuclear inclusions (NIIs) and the onset of behavioral abnormalities. The NES reduced levels of nuclear HprtQ150 protein in mouse brain and delayed both the presentation of NIIs and the onset of behavioral abnormalities. Together these results indicate the nucleus is the primary site of toxicity in HprtQ150 mice. Furthermore, the signals did not alter the relative regional distribution of NIIs, suggesting that factors other than nuclear access dictate the regional specificity of NII formation in this mouse model.

Appropriate Tissue- and Cell-specific Expression of a Single Copy Human Angiotensinogen Transgene Specifically Targeted Upstream of the HPRT Locus by Homologous Recombination

Development of experimental models by genetic manipulation in mice has proven to be very useful in determining the significance of particular genes in the development of or susceptibility to hypertension. Advances in molecular genetics, transgenic mouse technology, and physiological measurements in mice provided an opportunity to go a step further and develop models to analyze the physiological significance of specific gene variants potentially causing hypertension. In this report, we describe the development of a human angiotensinogen transgenic mouse model generated by targeting the human angiotensinogen gene upstream of the mouse HPRT locus by homologous recombination. The main benefit of this transgenic mouse model is that the human angiotensinogen gene is inserted into the mouse genome as a single copy at a predefined locus and in a specific orientation-a process that can be repeated utilizing other variants of this gene. We establish the validity of this approach by showing that the hAGT(hprt) mice have normal tissue- and cell-specific expression of the human angiotensinogen gene and normally produce and process the hAGT protein at physiological levels.

Toxicological evaluation and classification of the genotoxic, carcinogenic, reprotoxic and sensitising potential of tris(2-chloroethyl)phosphate

Tris(2-chloroethyl)phosphate is used as flame-proofing additive in polyurethane foam and paints. The substance was not mutagenic in the Salmonella/microsome test in 6 of 7 studies, two of which were performed on behalf of BG Chemie as part of its 'Programme for the Prevention of Health Hazards Caused by Industrial Substances'. No mutagenic potential has been detected in mammalian cells in vitro (mouse lymphoma test, reverse mutation and HPRT test). The substance produces no chromosome aberrations in vitro, but does cause questionable to significant increases in the sister chromatid exchange rate. Two in vivo micronucleus tests performed on behalf of BG Chemie have given no indications of clastogenicity, while a further test was inconclusive. A test involving Drosophila (interchromosomal mitotic recombination) was also negative. Overall, tris(2-chloroethyl)phosphate appears to have no genotoxic potential. According to the EU (European Union) classification criteria, the substance has not been classified as mutagenic in Germany. Summarising the existing studies on the carcinogenic potential of tris(2-chloroethyl)phosphate, kidney tumours as well as tumours of the Harderian gland were present at below the maximum tolerable dose, the kidney tumours arising in two different species (rats, mice) and in mice in two studies. These tumours do not spontaneously occur, and their incidence was also increased in comparison with historical control data. However, only at systemic toxic doses was the incidence of carcinomas of the kidney significantly increased. Below systemic toxic doses, only the benign kidney tumours were significantly increased, but this was dose-dependent. In conclusion, the data show a carcinogenic potential of tris(2-chloroethyl)phosphate, possibly with an epigenetic mechanism. A supporting indication is provided by two of three in vitro cell transformation tests with positive results. Therefore, the carcinogenicity has been classified as EU category C2 in Germany. Tris(2-chloroethyl)phosphate is not embryotoxic or teratogenic in rats with oral administration, even at doses that are maternally toxic, and does not impair postnatal development in the offspring. Accordingly, the developmental reproductive toxicity has not been classified in Germany. However, the substance adversely affects sperm and its motility with subchronic oral administration to rats and mice. Toxic effects on reproduction were seen in both generations in a 2-generation study in Swiss CD1 mice. The classification of the reproductive toxicity, endpoint fertility, has been suggested by BG Chemie to be RF2 according to the EU classification criteria, which is still under discussion and will be concluded by the time of the congress (in the meantime, the substance has been classified as RF2 in Germany.) There were no indications of skin-sensitising potential in a Buehler test in guinea pigs. Accordingly, no classification is necessary.

Development and utilization of the rat lymphocyte hprt mutation assay.

Much of the recent progress in the field of genetic toxicology has come from an increased understanding of the molecular and cellular biology of the mammalian organism. Most prominent has been the ability to detect and quantify somatic mutation and relate the nature of the mutation to the specific type of chemical damage. Building upon the foundation of the human lymphocyte hypoxanthine guanine phosphoribosyl transferase (hprt) system, and later, the mouse hprt system, methods for the detection and quantification of hprt mutations in rat lymphocytes were developed. These methods are described in this report as is the ongoing validation of the assay. Additionally, the characterization of the recovered mutants and a comparison of the mutation spectrum in the rat lymphocyte system to the spectrum in cancer genes, such as H-ras and p53, and the spectrum in transgenic systems, such as lacI, are included. The development of the rat lymphocyte hprt system and validation of the assay at the molecular level, provide an effective and reliable measure of genetic damage in an in vivo system which is readily comparable to measurement of genetic damage in the human.

DNA Methylation Patterns and Sequence Transitions of the CpG Island of Mouse Hprt during Early Embryogenesis

DNA Methylation Patterns and Sequence Transitions of the CpG Island of Mouse Hprt during Early Embryogenesis

Mosaic expression of an Hprt transgene integrated in a region of Y heterochromatin.

The sensitivity of small transgenes to position effects on their expression suggests that they could serve as indicators of the chromatin properties at their integration site. In particular, they might be expected to provide information on the functional properties of mammalian heterochromatin. We have produced a transgenic line that carries a mouse Hprt minigene on the Y chromosome. In situ hybridization localized the transgene to the heterochromatic portion of the Y. Analysis of transgene expression by isoelectric focusing indicated that the transgene is expressed in a mosaic pattern, and expressing cells have different levels of transgene activity. These findings can be explained as a position effect variegation induced by Y heterochromatin. However, two other transgenes, located at autosomal sites, also showed mosaic activity. If the mosaic transgene expression is attributed to the influence of the chromatin at the insertion site, the Y heterochromatin would appear less potent than some autosomal regions at inducing variegation. An alternative explanation consistent with our results is that the mosaic expression is a semi-autonomous characteristic of these transgene loci. Transgene-expressing and non-expressing cells differed in their ability to grow and be cloned in vitro, indicating that cellular differentiation affected the chromatin structure of the transgene locus on the Y. Karyotype analysis of male mice with the Y-linked transgene and from control male mice carrying the human HPRT transgene, or the mouse Pgk-1 gene at autosomal sites, indicated that the transgene-carrying Y is prone to non-disjunction, generating cells with two (or more) or no Y chromosomes in equal proportion. Further studies will determine if the propensity of this Y chromosome to mitotic errors is also observed in vivo.

A position- and orientation-dependent element in the first intron is required for expression of the mouse hprt gene in embryonic stem cells

The gene ( hprt) coding for mouse HPRT (hypoxanthine phosphoribosyltransferase) is transcribed from a promoter lacking CAAT and TATAA boxes. It is expressed ubiquitously, albeit at different levels, in all tissues and cultured cells. During investigations to characterise hprt transcription control elements required in embryonic stem (ES) cells and to develop compact hprt minigenes for gene-targeting strategies, we discovered a requirement for intron-1 sequences for expression in ES cells. The essential intron-1 element, which is 420 bp long, is located 230 bp downstream from the transcription start point and is shown to increase transcription from the hprt promoter in a position- and orientation-dependent manner. We propose that this element is an integral downstream part of the hprt promoter.

Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea.

We have determined the mutational spectrum of N-ethyl-N-nitrosourea (ENU) in exon 3 of the hypoxanthine (guanine) phosphoribosyltransferase gene (Hprt) in splenic T cells following in vivo exposure of male B6C3F1 mice (5-7 weeks old) to ENU. Hprt- mutants were isolated by culturing splenic T cells in microtiter dishes containing medium supplemented with interleukin 2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking Hprt exon 3. Identification of mutant sequences and purification of mutant DNA from contaminating wild-type Hprt DNA was accomplished by denaturing-gradient gel electrophoresis. Purified mutant DNA was then sequenced. Treatment of mice with ENU at 40 mg/kg of body weight produced a Hprt- mutant frequency of 7.3 x 10(-5) in splenic T cells, approximately 35-fold above background levels. Sixty-nine of the 521 Hprt- mutants analyzed contained mutations in exon 3 (13%). Transversions and transitions at A.T base pairs dominated the spectrum; 62 of the 69 exon 3 mutations were at A.T base pairs (14 different sites). Thirteen of 14 thymine bases undergoing mutation (61 of 62 mutations at A.T bases) were located on the nontranscribed strand of exon 3. The majority of the remaining mutations (6 of 69) were transitions at a single G.C base pair. These results suggest the importance of thymidine alkylation in ENU-induced mutagenesis in vivo. The mouse Hprt- T-cell cloning/sequencing assay described here may represent a useful system for studying the molecular mechanism of chemically induced mutation occurring in vivo in an endogenous gene.

The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat

In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase ( hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5′-phosphorirobsyl-1-pyrosphosphate (PRPP) and nuceotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.

A pgk:: hprt fusion as a selectable marker for targeting of genes in mouse embryonic stem cells: disruption of the T-cell receptor δ-chain-encoding gene

We have constructed a hypoxanthine phosphoribosyl transferase-selectable marker ( hprt) under the control of the phosphoglycerate kinase ( pgk) promoter. This construct permits cell growth in hypoxanthine/aminopterin/thymidine media and confers 6-thioguanine sensitivity upon mouse Hprt − embryonic stern cells, allowing either positive or negative selection in gene-targeting experiments. We have successfully targeted the gene encoding the T-cell receptor δ-chain using the pgkr. hprt fusion for counterselection.

Maturation-specific polyadenylation and translational control: diversity of cytoplasmic polyadenylation elements, influence of poly(A) tail size, and formation of stable polyadenylation complexes.

Early embryonic development in Xenopus laevis is programmed in part by maternally derived mRNAs, many of which are translated at the completion of meiosis (oocyte maturation). Polysomal recruitment of at least one of these mRNAs, G10, is regulated by cytoplasmic poly(A) elongation which, in turn, is dependent upon the cytoplasmic polyadenylation element (CPE) UUUUUUAUAAAG and the hexanucleotide AAUAAA (L. L. McGrew, E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter, Genes Dev. 3:803-815, 1989). We have investigated whether sequences similar to the G10 RNA CPE that are present in other RNAs could also be responsible for maturation-specific polyadenylation. B4 RNA, which encodes a histone H1-like protein, requires a CPE of the sequence UUUUUAAU as well as the polyadenylation hexanucleotide. The 3' untranslated regions of Xenopus c-mos RNA and mouse HPRT RNA also contain U-rich CPEs since they confer maturation-specific polyadenylation when fused to Xenopus B-globin RNA. Polyadenylation of B4 RNA, which occurs very early during maturation, is limited to 150 residues, and it is this number that is required for polysomal recruitment. To investigate the possible diversity of factors and/or affinities that might control polyadenylation, egg extracts that faithfully adenylate exogenously added RNA were used in competition experiments. At least one factor is shared by B4 and G10 RNAs, although it has a much greater affinity for B4 RNA. Additional experiments demonstrate that an intact CPE and hexanucleotide are both required to compete for the polyadenylation apparatus. Gel mobility shift assays show that two polyadenylation complexes are formed on B4 RNA. Optimal complex formation requires an intact CPE and hexanucleotide but not ongoing adenylation. These data, plus additional RNA competition studies, suggest that stable complex formation is enhanced by an interaction of the trans-acting factors that bind the CPE and polyadenylation hexanucleotide.

Maturation-Specific Polyadenylation and Translational Control: Diversity of Cytoplasmic Polyadenylation Elements, Influence of Poly(A) Tail Size, and Formation of Stable Polyadenylation Complexes

Early embryonic development in Xenopus laevis is programmed in part by maternally derived mRNAs, many of which are translated at the completion of meiosis (oocyte maturation). Polysomal recruitment of at least one of these mRNAs, G10, is regulated by cytoplasmic poly(A) elongation which, in turn, is dependent upon the cytoplasmic polyadenylation element (CPE) UUUUUUAUAAAG and the hexanucleotide AAUAAA (L. L. McGrew, E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter, Genes Dev. 3:803-815, 1989). We have investigated whether sequences similar to the G10 RNA CPE that are present in other RNAs could also be responsible for maturation-specific polyadenylation. B4 RNA, which encodes a histone H1-like protein, requires a CPE of the sequence UUUUUAAU as well as the polyadenylation hexanucleotide. The 3' untranslated regions of Xenopus c-mos RNA and mouse HPRT RNA also contain U-rich CPEs since they confer maturation-specific polyadenylation when fused to Xenopus B-globin RNA. Polyadenylation of B4 RNA, which occurs very early during maturation, is limited to 150 residues, and it is this number that is required for polysomal recruitment. To investigate the possible diversity of factors and/or affinities that might control polyadenylation, egg extracts that faithfully adenylate exogenously added RNA were used in competition experiments. At least one factor is shared by B4 and G10 RNAs, although it has a much greater affinity for B4 RNA. Additional experiments demonstrate that an intact CPE and hexanucleotide are both required to compete for the polyadenylation apparatus. Gel mobility shift assays show that two polyadenylation complexes are formed on B4 RNA. Optimal complex formation requires an intact CPE and hexanucleotide but not ongoing adenylation. These data, plus additional RNA competition studies, suggest that stable complex formation is enhanced by an interaction of the trans-acting factors that bind the CPE and polyadenylation hexanucleotide.