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An experiment was conducted to determine the effect of feeding reduced crude protein (CP) diets to Ross × Ross 708 male broilers while providing adequate essential amino acid (AA) concentrations on growth performance, nitrogen (N) and ammonia output, and carcass characteristics from d 1 to 33 post hatch. Birds received 1 of 6 dietary treatments (10 replicate pens per treatment) varying in CP content. Diet 1 (control) was formulated with DL-Met, L-Lys, and L-Thr (23.2, 20.7, and 19.1% CP) in the starter (1-14 d of age), grower (15-25 d of age), and finisher (26-33 d of age) periods, respectively. Dietary L-Val, Gly (only in starter period), L-Ile, L-Arg, and L-Trp were sequentially supplemented in the order of limitation in Diets 2 through 6. Dietary CP was reduced gradually across the dietary treatments resulting in a CP reduction in Diets 1 to 6 by 3.4, 3.4, and 2.3% points in the starter, grower, and finisher periods, respectively. At d 14, 25, and 33 posthatch, feed conversion decreased (P < 0.05) with L-Val addition (Diet 2) and increased (P < 0.01) with L-Val to L-Trp addition (Diet 6) to the control. Dietary treatments did not alter weights and yields of carcass, breast, drum, or thighs. Dietary CP reduction with added L-Val (Diet 2), L-Val to L-Arg (Diet 5), or L-Val to L-Trp (Diet 6) increased abdominal fat (P < 0.01) compared with control. Nitrogen excretion (g/bird; P = 0.003) and equilibrium ammonia concentration (mg/kg; P = 0.041) at day 33 reduced by 16% and 48% respectively in birds fed reduced-CP diets with L-Val to L-Trp (Diet 6) compared with control-fed birds. This study indicated that sequential addition of supplemental AA in the order of limitation from DL-Met to L-Arg allowed reduction of dietary CP beyond 2%-point without depressing growth performance and meat yield of broilers from day 1 to 33 while reducing nitrogen excretion and ammonia emissions., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

There is a gap in the understanding of the relationship between dietary phytate levels and the relative efficacy of phytase to improve amino acid (AA) digestibility in pigs and chickens. Two experiments were conducted to investigate the effect of exogenous phytase on standardized ileal digestibility (SID) of AA and the apparent ileal digestibility (AID) of P in both standard- (SP) and high-phytate (HP) diets for broilers and swine. There were either 40 cages of Cobb 500 male broilers or 10 crossbred barrows (35 kg) fitted with ileal T-cannulas. Both studies were allotted to five dietary treatments (8 replicates). Treatments consisted of four corn-soybean meal-based diets arranged in a 2 × 2 factorial of standard or high phytate and exogenous phytase at 0 or 1 000 phytase units (FYT)/kg; and one N-free diet. Birds were fed a common starter diet from d 0 to 20 and fed experimental diets from d 20 to 25. Birds were euthanized on d 25 via CO 2 asphyxiation, and digesta were collected from the terminal ileum. Pigs were fed for a total of four 7-d periods, where digesta were collected on d 6 and 7 of each period. Diet and digesta samples were analyzed for DM, N, Ti, AA, and P to determine AA and P digestibility. The SID of AA was determined by correcting the AID of AA for the basal endogenous losses estimated using the N-free diet. Main effects of the diet type (standard or HP) and phytase (0 or 1 000 FYT/kg), and the interaction of diet type and phytase were evaluated. For both experiments, the HP diets produced lower SID of AA compared to the SP (P < 0.001). For broilers, there was a phytase effect (P < 0.001) for the SID of all AAs evaluated regardless of the diet type. For pigs, phytase improved (P < 0.05) the SID of Met, Lys, Cys, Glu and Ser and tended to improve (P < 0.10) Arg, Leu, Thr, and Tyr. There were no significant interactions for either experiment. For both experiments, AID of P was lower for the HP diets (P < 0.01), and phytase produced greater AID of P for both diet types (P < 0.01). These data indicate that phytase greatly improves the digestibility of P for broilers and pigs and has the ability to significantly increase the digestibility of amino acids for these animals, regardless of the dietary phytate P., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Two experiments (Exp.) were conducted to validate a 3-point model for the regression method of determining ME, using canola meal (CM) and wheat as test ingredients (TI). Corn-soybean meal-based test diets (TD) contained 0, 100, 200, or 300 g/kg CM, added at the proportional expense of all energy contributing ingredients for Exp. 1, and 0, 150, 300, or 450 g/kg wheat for Exp. 2. For each Exp., 192 Cobb 500 male broiler chickens were weighed and allotted by BW to 1 of 4 treatments at d 21 post hatching in a randomized complete block design. Growth performance and metabolizability responses were evaluated for linear and quadratic effects using orthogonal contrasts, and ileal digestible energy (IDE), ME, and MEn of TI were determined by regressing the TI-associated energy against the dry matter intake of TI using a generalized linear model. Four data sets were used to determine ME, using all possible 3 and 4-point combinations of TD in each Exp. Increasing TI inclusion elicited linear decreases (P < 0.01) in the digestibility and metabolizability of DM and GE in the 2 studies. The ME of CM obtained from the 4 data sets ranged from 1,731 to 1,992 kcal/kg DM, however, excluding the highest concentration of CM produced the highest estimate of ME, whereas the other 3 sets ranged from 1,731 to 1,793 kcal/kg DM. The ME of wheat from the 4 data sets had a smaller range of 3,041 to 3,106 kcal/kg DM. Excluding the highest concentration of either TI produced higher standard errors for the estimate of ME compared to the other 3 sets (42 and 36% greater SE, respectively). Results for IDE and MEn were similar. These data indicate that there is no difference in the variation of estimates between the 3 and 4-point models, provided that the inclusion of the TI is adequate and both models represent the linearity and variability of responses., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Three experiments (Exp) were conducted to determine optimal digestible Ile to Lys ratios for male Yield Plus × Ross 708 broilers from approximately 1.0 to 4.0 kg BW. Broilers were fed dose-response diets with inclusions of blood cells that were formulated to contain a gradient of digestible Ile to Lys ratios (0.46 to 0.83). Treatments for Exp 1 to 3 were fed from 21 to 35, 28 to 42, and 35 to 49 d of age, respectively, to target market weights from 2.5 to 4.0 kg. Experiments utilized positive control (PC) diets that did not contain blood cells and were formulated to the same Ile ratios as Treatment 5. Birds and feed were weighed by pen on the first and last days of the experimental period to determine growth performance. Selected broilers were processed and deboned to determine carcass characteristics. For all Exp, quadratic effects (P ≤ 0.001) were observed with BW gain, feed conversion ratio (FCR), breast meat weight, and breast meat yield (BMY) as digestible Ile to Lys ratios increased. Contrasts between PC and Treatment 5 for each Exp displayed no effect of blood cell inclusion with the exception of FCR in Exp 1 (P = 0.001) and BMY in Exp 3 (P = 0.017). Optimum digestible Ile to Lys ratios for Exp 1 were determined to range from 0.640 to 0.725 for growth from 1.0 to 2.5 kg BW (P ≤ 0.001) and breast meat characteristics. In Exp 2, optimum ratios ranged from 0.664 to 0.682 for growth and breast meat characteristics from 1.6 to 3.1 kg BW (P ≤ 0.001). For growth and breast meat characteristics of broilers in Exp 3, optimum ratios ranged from 0.625 to 0.730, from 2.6 to 3.9 kg BW (P ≤ 0.001). Based on these findings, optimum digestible Ile to Lys ratios were determined to range from 0.63 to 0.73 for broilers from 1.0 to 4.0 kg BW., Competing Interests: DISCLOSURES The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Fragile X syndrome (FXS) is an inherited form of intellectual disability that is usually caused by expansion of a polymorphic CGG repeat in the 5' untranslated region of the X-linked FMR1 gene, which leads to hypermethylation and transcriptional silencing. Two non-neurological phenotypes of FXS are enlarged testes and connective tissue dysplasia, which could be caused by alterations in a growth factor signaling pathway. FXS patients also frequently have autistic-like symptoms, suggesting that the signaling pathways affected in FXS may overlap with those affected in autism. Identifying these pathways is important for both understanding the effects of FMR1 inactivation and developing treatments for both FXS and autism. Here we show that decreasing the levels of the insulin-like growth factor (Igf) receptor 1 corrects a number of phenotypes in the mouse model of FXS, including macro-orchidism, and that increasing the levels of IGF2 exacerbates the seizure susceptibility phenotype. These results suggest that the pathways altered by the loss of the FMR1-encoded protein (FMRP) may overlap with the pathways affected by changes in Igf signaling or that one or more of the proteins that play a role in Igf signaling could interact with FMRP. They also indicate a new set of potential targets for drug treatment of FXS and autism spectrum disorders., (© 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Mammals with excess insulin-like growth factor 2 (IGFII) during embryogenesis have developmental defects that can lead to perinatal lethality. In adults, higher levels of IGFII increase the risk of cancer and may accelerate the development of atherosclerosis. IGFII can be increased as a consequence of genetic abnormalities and polymorphisms, and through epigenetic mechanisms. Decreasing IGFII levels thus can benefit human health. Degradation of IGFII is mediated by the insulin-like growth factor type 2 receptor (IGF2R). The growth-stimulatory effects of IGFII, and their attenuation by the IGF2R, are considered important for the evolution of IGFII/IGF2R interaction and imprinting. The IGFII/IGF2R interactions during development have been previously examined in mice carrying knock-out alleles of these genes or their regulators. Here we tested the ability of the IGF2R to ameliorate the negative effects of IGFII on development and survival in crosses between Igf2 and Igf2r transgenic mice, which may be a better model for natural variations in the levels of these genes' products. A fraction of hemizygous Igf2 transgenic mice die in the perinatal period, some with cleft palates, with an ensuing reduction in the frequency of transgenic mice among the surviving offspring. The Igf2r transgene lowers the frequency of cleft palate and increases the percentage of Igf2 transgenic mice among the live offspring. These findings draw attention to the fact that Igf2-associated lethality selects for the retention of IGFII/IGF2R binding in present day mammals; it may have played a similar role in the acquisition of IGFII/IGF2R binding in ancient mammals.

High-dose inhaled tobramycin has been increasingly used for treatment and suppression of Pseudomonas aeruginosa pulmonary infections, especially in patients with cystic fibrosis. The advantage of inhalation over other routes of administration is minimal systemic absorption, which reduces the potential for adverse effects. However, cases of adults who had elevated serum concentrations and experienced systemic adverse effects due to excessive systemic absorption after inhaled tobramycin have been reported. We describe a prematurely born infant with numerous congenital and acquired disorders who required assisted mechanical ventilation and a 60-day stay in the neonatal intensive care unit (NICU). Tracheostomy and mechanical ventilatory support were required throughout the infant's hospital stay. The patient developed several pulmonary infections caused by various bacteria. He was treated with multiple antibiotics, including two different dose preparations of inhaled tobramycin 80 mg and 300 mg, administered through the tracheostomy and the ventilator. The infant was given a total of five preparations of tobramycin 80 mg/dose and three of 300 mg/dose, for a total cumulative dose of 1,300 mg over a 6-day period. His tobramycin concentrations increased, prompting discontinuation of the inhaled tobramycin. The infant died on day 60. To our knowledge, this is the first report of elevated tobramycin concentrations after inhalation in an infant. Although studies have found that tobramycin is safe and effective, certain patient populations are more at risk for toxicity. Tobramycin concentrations should be closely monitored in patients with significant underlying renal disorders, especially those in age-group extremes.

The insulin-like growth factor-II (IGF-II) receptor (IGF2R) regulates the level or activity of numerous proteins, including factors that control growth and differentiation. Frequent loss or inactivation of this receptor in a diverse group of tumors indicates that it may act as a tumor suppressor, but it is not known which functions of this receptor are selected against in the tumors. Lysosomal targeting and degradation of the growth-promoting IGF-II has been proposed as a mechanism for the tumor suppressor effects of IGF2R. As a genetic test of this hypothesis in vivo, we have produced Igf2r transgenic mice that ubiquitously express the transgene and have crossed these mice with mice that develop mammary tumors as a consequence of Igf2 overexpression. Our findings indicate that the presence of the Igf2r transgene delays mammary tumor onset and decreases tumor multiplicity in Igf2 transgenic mice. These findings are relevant to human tumors and preneoplastic conditions accompanied by altered IGF2 expression.

The radiation-induced mutation Oligosyndactylism (Os) is associated with limb and kidney defects in heterozygotes and with mitotic arrest and embryonic lethality in homozygotes. We reported that the cell cycle block in Os and in the 94-A/K transgene-induced mutations is due to disruption of the Anapc10 (Apc10/Doc1) gene. To understand the genetic basis of the limb and kidney abnormalities in Os mice we characterized the structural changes of chromosome 8 associated with this mutation. We demonstrate that the Os chromosome 8 has suffered two breaks that are 5 cM ( approximately 10 Mb) apart and the internal fragment delineated by the breaks is in an inverted orientation on the mutant chromosome. While sequences in proximity to the distal break are present in an abnormal Os-specific Anapc10 hybrid transcript, transcription of these sequences in normal mice is low and difficult to detect. Transfer of the Os mutation onto an FVB/N background indicated that the absence of dominant effects in 94-A/K mice is not due to strain background effects on the mutation. Further analysis of this mutation will determine if a gene interrupted by the break or a long-range effect of the rearrangement on neighboring genes is responsible for the dominant effects of Os.

Foreign DNA injected into mouse embryos integrates into the host chromosomes and is usually transmitted stably to the progeny. Rare cases of transgene instability have been described, and these can help our understanding of the rules that govern the organization and stability of endogenous DNA. We have observed unusual inheritance in three transgenic lines produced with a partially in vitro methylated Igf2 construct. All three founders transmitted to their progeny two different transgene patterns, A and B. Pattern A was inherited in accordance with expectation, whereas pattern B was associated with several abnormal characteristics, including fewer than expected transgenic progeny, evidence for instability and loss from the somatic tissues of some of the progeny, and high incidence of runting and perinatal death that did not appear correlated with transgene retention. The absence of these features in transgenic mice produced with the unmethylated version of the same construct indicated that prior methylation played a role in the unusual behavior of these transgenes. We hypothesize that patterns A and B were formed by transgenes that differed in their methylation, and that pattern B methylation led to instability of the transgene locus. Runting and early lethality in the pattern B sublines may be the result of transgene rearrangements, which result in transgene amplification with adverse effects of increased IGFII dosage, and/or deletions, which may affect endogenous genes required for viability. These findings provide further evidence that DNA methylation plays a role in genome stability and indicate that perturbations in the normal pattern of methylation may have destabilizing effects that extend through several generations.

Oligosyndactylism (Os) is a radiation-induced mouse mutation associated with recessive lethality and a dominant effect on limb and kidney development. The lethal effect of the mutation is due to a cell-autonomous block in the transition from metaphase to anaphase. We have previously characterized two transgene-induced mutations, 94-A and 94-K, which are allelic with Os. These mutations facilitated the identification of genomic segments and transcribed sequences in the affected region. One of the transcripts in this region corresponds to the mouse homolog of the anaphase-promoting complex component APC10/DOC1. The disruption of this gene can explain the mitotic arrest phenotype of all three alleles of Os., (Copyright 2001 Academic Press.)

CpG islands are GC-rich regions located in the promoter regions of housekeeping genes and many tissue-specific genes. While most CpG islands are normally unmethylated, island methylation can occur and is associated with silencing of the corresponding gene. Experiments with transgenic mice and DNA transfection in pluripotential embryonic cells have led to the conclusion that the information required for protecting the islands from methylation is contained within the CpG islands themselves and have identified Sp1 binding sites as an important element in establishing and/or maintaining the methylation-free state of CpG islands. To examine the generality of these observations, we analyzed the methylation of one of the mouse Igf2 CpG islands and its flanks in transgenic mice. We observed that the undermethylated state of this region is dependent on the presence of a separate cis-regulatory element, the H19 enhancers. These tissue-specific enhancers had a ubiquitous, non-tissue-specific effect on island region methylation. Structural alterations outside of the island and these enhancers also affected this region's methylation. These findings indicate that the methylation of some CpG island-containing regions is more sensitive than previously believed to the activity of distant cis-regulatory elements and to structural alterations in nonisland sequences in cis., (Copyright 1999 Academic Press.)

The insulin-like growth factor II is mitogenic for a number of cell types and can inhibit apoptosis. The frequent expression of this gene in human and experimental animal tumors indicates that insulin-like growth factor-2 may play an important role in tumor development. It has also been hypothesized that overexpression of this growth factor may be responsible for the increased incidence of childhood tumors in patients with Beckwith-Wiedemann syndrome. To assess the effects of Igf2 on tumor development we produced six transgenic mouse lines that express the gene under the control of the H19 enhancers. Transgenic expression was initiated in the embryonic period but remained high in several adult tissues, including the mammary gland, lung, and liver. Adult transgenic females from five of the transgenic lines developed often multiple mammary tumors that had the ability to metastasize. Increased incidence of other solid tumors was also noted in older mice. These findings indicate that Igf2 expression increases the probability of malignant transformation and that the mammary gland is at a particularly high risk of tumor development in response to chronic increase in Igf2 gene dosage.

The insulin-like growth factor II (IGFII) is a mitogen for a number of cell types in vitro and is required for normal embryonic growth. It has been hypothesized that overexpression of IGF2 is responsible for the increased growth and tumor predisposition in patients with Beckwith-Wiedemann syndrome. Association of increased levels of IGFII with increased growth is also incorporated in a current model for the evolution of Igf2 imprinting. Different experimental approaches to increasing IGFII levels in the mouse have yielded different results with respect to its effects on growth, viability, and tumor development. To investigate the consequences of IGf2 overexpression in the embryonic period, without alterations in the activity of other genes, we produced transgenic mice that express the Igf2 gene under the control of the H19 enhancers. Transgene expression in the embryonic period had no significant effect on the overall size of the embryos, but was associated with perinatal lethality in homozygous, and some heterozygous, mice. A large fraction of homozygous mice also developed a cleft palate. These findings indicate that overexpression of Igf2 can have an adverse effect on viability in the absence of a pronounced effect on overall body growth. The results are consistent with the view that growth and perinatal viability are affected differently by Igf2 overexpression in endodermal and mesodermal tissues.

Oligosyndactylism (Os) is a radiation-induced mutation on mouse chromosome 8 associated with early postimplantation lethality in homozygotes and abnormal development of the limbs and kidneys in heterozygotes. The recessive lethal effect of Os is due to a mitotic block of the embryonic cells that becomes apparent at the blastocyst stage, but it is not known if the heterozygous effect of Os is due to haploinsufficiency of the gene responsible for the mitotic arrest, or is due to mutation(s) of other gene(s). We have recently described a transgene-induced recessive mutation, 94-A/K, that results in early postimplantation death of the embryos, and we have mapped this mutation to the same region of chromosome 8 where Os has been assigned. On the basis of complementation tests between transgenic and Os/+ mice, in vitro growth characteristics and increased mitotic index of 94-A/K embryos, and molecular structural analysis of 94-A and 94-K transgenic and Os/+ mice, we conclude that the 94-A/K mutation represents a new allele of Os. This insertional mutation should facilitate the isolation of a mammalian gene essential for normal progression of the cell cycle beyond metaphase.

We have produced three lines of transgenic mice that contain additional copies of the mouse phosphoglycerate kinase 1 (Pgk1) gene. Two of these lines, 94-A and 94-K, which are descendants of a common founder, did not produce liveborn progeny carrying two copies of these transgenes (i.e., A/A, K/K, or A/K). Genotyping of midgestation embryos showed that A/K embryos are dead by Embryonic Day 10. Comparison of the level of transgene expression in the three transgenic lines ruled out PGK1 toxicity as the cause of death of A/A, A/K, and K/K embryos. The death of A/A, K/K, and A/K transgenic mice was therefore attributed to an insertional mutation disrupting a gene or genes essential for normal embryogenesis. Analysis of the structure of the 94-A and 94-K transgenes indicated that they differ in the number of tandem repeats and in the positions of the transgene-cellular DNA junctions. To determine if the two transgenes represent a single integration followed by a rearrangement or two independent integration events, we cloned the endogenous sequences surrounding the 94-A and 94-K transgene insertion sites. Restriction analysis of the isolated genomic clones indicated that the endogenous sequences abutting the 3' ends of the 94-A and 94-K transgenes are separated by less than 20 kb, providing strong support for the single integration model. Further analysis indicated that the 94-A transgene is associated with a deletion of at least 18 kb and is located in the vicinity of a widely transcribed endogenous gene. Chromosomal mapping of the endogenous sequences flanking the 94-A and 94-K transgene insertions using mouse-hamster somatic cell hybrids and a (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei backcross panel allowed us to assign the 94-A(K) transgene insertion to the subcentral region of mouse chromosome 8.

Homeobox genes regulate multiple aspects of mouse development including the axial patterning in the central nervous system. Some of the more divergent members of this gene family are expressed in the anterior region of the central nervous system where the Hox genes are not expressed. We previously reported the isolation of a mouse homeobox gene, Dbx, from the forebrain. Here we describe its genomic structure, complete cDNA sequence, characterization of the basal promoter and the expression pattern at different stages of the developing embryo. During early and mid-gestation, Dbx expression is restricted to the telencephalon, diencephalon, dorsal mesencephalon and spinal cord. At later gestational stages, Dbx expression continues in the dorsal mesencephalon and diencephalon, in which expression is more restricted than at the earlier stages. Dbx transcripts were also detected in the primitive cerebellum. At all stages, the Dbx expressing regions contain a high proportion of proliferating cells, suggesting a role for Dbx in patterning the central nervous system during embryogenesis.

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.

Following the discovery of the homeobox as a conserved sequence in developmentally important genes of Drosophila, a plethora of such sequences have been identified in evolutionarily distant organisms. Among mammals, the mouse homeobox genes have been studied most intensively with a hope of deciphering basic mechanisms of embryonic development. The genomic arrangement of many mouse homeobox genes is similar to the organization of the Drosophila genes, suggesting that they arose as a consequence of gene duplication and divergence from a primordial cluster during evolution. Homeobox genes encode proteins that may form a part of the autoregulatory and transregulatory network specifying positional value in the embryo. Supporting this view, the more diverged members of this growing family function as transcription factors, some of which regulate the expression of tissue-specific genes. Mouse homeobox genes are expressed during embryonic development in a spatially restricted manner and alterations in their expression pattern can disrupt embryonic development. The implications of these findings will be discussed in the context of the role of homeobox genes in the embryonic development of Drosophila and other organisms.

Previous studies on V79 Chinese hamster cells have shown that bromodeoxyuridine (BrdU) -resistant variants deficient in thymidine kinase (TK) activity arise by a multistep process which is initiated by a random event and progresses gradually during serial culture in the presence of the drug. In order to determine the molecular basis for the loss of TK activity in these cells, the TK gene was isolated from a lambda phage library of genomic V79 DNA, using a fragment of the human TK gene as a probe. One phage isolated contained the entire TK gene in a 15-kb insert, as demonstrated by the ability of the phage DNA to transform Ltk- mouse cells to the TK+ phenotype. Five fragments spanning the entire gene were then subcloned into the plasmid pUC12 for DNA methylation studies. With these probes it was shown by hybridization analysis that the copy number of the TK gene in V79 cells is about four times the copy number in CHO cells and Chinese hamster liver cells. Southern hybridization analysis of the DNA from first-stage variants partially resistant to BrdU indicated that partial resistance was accompanied by deletion of a number of copies of the TK gene in V79 cells. However, the subsequent gradual transition to full BrdU resistance and full loss of TK activity was correlated with a gradual hypermethylation of sites in the 5' region of the TK gene, with no further change in gene copy number.