BSHI Guideline: HLA matching and donor selection for haematopoietic progenitor cell transplantation.

A review of the British Society for Histocompatibility and Immunogenetics (BSHI) "Guideline for selection and HLA matching of related, adult unrelated donors and umbilical cord units for haematopoietic progenitor cell transplantation" was undertaken by a BSHI appointed writing committee. Literature searches were performed, and the data extracted were presented as recommendations according to the GRADE nomenclature.

HLA-DRB1*03:49, a novel allele identified by group-specific sequence-based typing in a North European individual.

Identification of the antigen presenting molecule HLA-DRB1*03:49 by group-specific sequence-based typing.

In situ Raman spectroscopy of H2 interaction with WO3 films.

It is well known that WO(3) interacts efficiently with H(2) gas in the presence of noble metals (such as Pd, Pt and Au) at elevated temperatures, changing its optical behaviors; and that its crystallinity plays an important role in these interactions. For the first time, we investigated the in situ Raman spectra changes of WO(3) films of different crystal phases, while incorporating Pd catalysts, at elevated temperatures in the presence of H(2). The Pd/WO(3) films were prepared using RF sputtering and subsequently annealed at 300, 400 and 500 °C in air in order to alter the dominant crystal phase. The films were then characterized using SEM, XRD, XPS, and both UV-VIS and Raman spectroscopy. In order to fundamentally study the process, the measurements were conducted when films were interacting with 1% H(2) in synthetic air at elevated sample temperatures (20, 60, 100 and 140 °C). We suggest that the changes of Raman spectra under such conditions to be mainly a function of the crystal phase, transforming from monoclinic to a mix phase of monoclinic and orthorhombic achieved via increasing the annealing temperature. The as-deposited sample consistently shows similar Raman spectra responses at different operating conditions upon H(2) exposure. However, increasing the annealing temperature to 500 °C tunes the optimum H(2) response operating temperature to 60 °C.

Molecular control of mitochondrial function in developing rhesus monkey oocytes and preimplantation-stage embryos.

The mitochondrion undergoes significant functional and structural changes, as well as an increase in number, during preimplantation embryonic development. The mitochondrion generates ATP and regulates a range of cellular processes, such as signal transduction and apoptosis. Therefore, mitochondria contribute to overall oocyte quality and embryo developmental competence. The present study identified, for the first time, the detailed temporal expression of mRNAs related to mitochondrial biogenesis in rhesus monkey oocytes and embryos. Persistent expression of maternally encoded mRNAs was observed, in combination with transcriptional activation and mRNA accumulation at the eight-cell stage, around the time of embryonic genome activation. The expression of these transcripts was significantly altered in oocytes and embryos with reduced developmental potential. In these embryos, most maternally encoded transcripts were precociously depleted. Embryo culture and specific culture media affected the expression of some of these transcripts, including a deficiency in the expression of key transcriptional regulators. Several genes involved in regulating mitochondrial transcription and replication are similarly affected by in vitro conditions and their downregulation may be instrumental in maintaining the mRNA profiles of mitochondrially encoded genes observed in the present study. These data support the hypothesis that the molecular control of mitochondrial biogenesis, and therefore mitochondrial function, is impaired in in vitro-cultured embryos. These results highlight the need for additional studies in human and non-human primate model species to determine how mitochondrial biogenesis can be altered by oocyte and embryo manipulation protocols and whether this affects physiological function in progeny.

X chromosome imprinting and inactivation in the early mammalian embryo.

Quantitative differences in X-linked gene expression between androgenetic (two paternal genomes), gynogenetic (two maternal genomes) and normal embryos provide clues into the roles of genomic imprinting and the X:autosome ratio in controlling X chromosome function during development. These data and many others can be accounted for by a new model of X-chromosome-inactivation (XCI). Expression of the Xist RNA from all paternal X chromosomes during development preimplantation leads to repression of genes near the X-chromosome-inactivation center (Xic). Other genes are repressed as a result of spreading of the inactivation, but only in embryos with at least two X chromosomes. XY androgenones are only deficient in expression of genes near the Xic and can form blastocysts, whereas XX androgenones completely inactivate both X chromosomes and die before the blastocyst stage. The X:autosome ratio regulates XCI solely by promoting the spread of inactivation away from the Xic on chromosomes that express Xist. Methylation of the maternal Xist gene is retained in extraembryonic tissues, so that gynogenones and parthenogenones cannot express Xist, do not undergo XCI in those tissues, and so have extraembryonic defects. This model should be relevant to understanding how aberrant X chromosome regulation might occur and how this might contribute to distortion of the X-chromosome-transmission ratio, sex ratio distortion, and disease.

Thyroid hormonelike actions of 3,3',5'-L-triiodothyronine nad 3,3'-diiodothyronine.

l-Thyroxine is converted to 3,5,3'-l-triiodothyronine (T(3)) as well as to 3,3',5'-l-triiodothyronine (reverse T(3)). One product of further deiodination is 3,3'-diiodothyronine (3,3'T(2)). The serum levels of reverse T(3) and 3,3'T(2) change considerably in various physiological and disease states. We previously found that reverse T(3) and 3,3'T(2) bind to the solubilized hepatic nuclear "receptors" for thyroid hormones. This led us to study binding and actions of these metabolites in cultured rat pituitary cells in which glucose consumption and growth hormone production are regulated by T(3) and l-thyroxine. Reverse T(3) and 3,3'T(2) stimulated growth hormone production and glucose consumption and inhibited nuclear binding of radioactive T(3). Either metabolite produced maximal effects that equaled those of T(3), and neither inhibited the T(3) response. Further, additive effects were observed when reverse T(3) was combined with submaximal concentrations of T(3). In serum-free and serum-containing media, concentrations of 3,3'T(2) 50- to 70- and 10- to 100-fold greater, respectively, than those of T(3) were required for equivalent stimulations and for inhibition of nuclear binding by T(3). The relative activity differences under the two conditions can be attributed to weaker serum protein binding of 3,3'T(2) than T(3). With cells in serum-free media, reverse T(3) was a less avid competitor than 3,3'T(2) for T(3) binding by the nuclear receptors, and was less potent than 3,3'T(2) (0.001 the potency of T(3)) in inducing growth hormone production or glucose oxidation. In incubations with serum-containing media, reverse T(3) was an ineffective competitor for T(3) binding, and had only 0.1 the inducing potency of 3,3'T(2) (0.001 the potency of T(3)). The weaker activity of reverse T(3) relative to 3,3'T(2) in serum-containing media could be explained by stronger serum binding of reverse T(3) than 3,3'T(2). In addition, after long-term incubation of cells with radioactive reverse T(3), much of the cell-associated radioactivity was recovered as 3,3'T(2). These studies suggest that reverse T(3) and 3,3'T(2) can stimulate thyroid hormone-regulated functions as weak agonists by acting via the same receptors that mediate T(3) actions. Moreover, some of the effects of reverse T(3) may be due to 3,3'T(2) produced by deiodination of reverse T(3).

Mitogen stimulation affects contractile protein mRNA abundance and translation in embryonic quail myocytes.

In cultures of differentiated, fusion-blocked muscle cells obtained from embryonic Japanese quail (Coturnix coturnix japonica), mitogen stimulation leads to an immediate reduction in the rates of synthesis of skeletal muscle myosin heavy chain (MHC) and alpha-actin. The molecular mechanisms responsible for this downregulation were examined. The cellular abundances of the alpha-actin and MHC mRNAs were affected differently by mitogen stimulation; alpha-actin mRNA abundance declined by an amount which quantitatively accounted for the observed decrease in alpha-actin synthesis, whereas MHC mRNA abundance remained virtually unchanged during the first 6 h following mitogen stimulation, a period during which MHC synthesis declined by more than 70%. MHC mRNA abundance did decline between 6 and 12 h after mitogen stimulation. Downregulation of MHC synthesis therefore involves an initial block in mRNA translation combined with a later loss of MHC mRNA from the cytoplasma, while alpha-actin synthesis is regulated at the level of mRNA abundance. These observations are consistent with the hypothesis that, in addition to transcriptional activation of muscle-specific genes, skeletal muscle differentiation normally involves cell cycle-dependent modulations in cellular factors which control message stability and message translation.

Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases.

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.

Examination of the oncogenic potential of a tumor-associated antigen, intestinal alkaline phosphatase, in HeLa x fibroblast cell hybrids.

An exclusive correlation exists between the ectopic expression of the cell-surface marker, intestinal alkaline phosphatase (IAP), and the tumorigenic phenotype of segregants derived from suppressed, nontumorigenic HeLa x fibroblast cell hybrids. This specific association suggests that loss of tumor suppressor function is closely linked to the re-expression of IAP and, therefore, that IAP may be a critical oncogenic factor in these cells. To address this directly, we have used a HeLa IAP cDNA expression vector (pHIAP) to introduce constitutive IAP expression into a nontumorigenic HeLa x fibroblast cell hybrid. Sequence analysis of the HeLa IAP cDNA revealed 5 separate nucleotide alterations when compared to the native IAP cDNA sequence; however, none of these resulted in amino acid substitutions. Four pHIAP transfectants were analyzed for the presence of the intact integrated IAP cDNA and their relative expression levels of exogenous IAP mRNA and protein. The functional integrity of the cDNA-derived IAP product was confirmed by demonstrating proper enzymatic activity and localization to the extracellular membrane. The tumorigenic potentials of the pHIAP transfectants were assayed by s.c. injection into athymic nude mice. No tumors were observed, even after an 11-week incubation in animals. Therefore, expression of IAP in the nontumorigenic HeLa x fibroblast cell hybrid is not sufficient to confer the tumorigenic phenotype. Although ectopic IAP expression is unlikely to be functionally relevant to tumorigenicity in these hybrids, the significance of IAP as a tumor marker is still evident from its apparent strong association with a tumor suppressor locus.

Effect of 3,5,3'-triiodo-L-thyronine on the incidence and growth kinetics of spontaneous mammary tumors in C3H/HeN mice.

The present study compares the incidence and growth kinetics of spontaneous mammary tumors in 3,5,3'-triiodo-L-thyronine (T3)-treated (0.7 microgram/day, i.p.) C3H/HeN retired breeder mice with tumors occurring spontaneously in this strain (0.15 M NaCl, i.p.). T3 treatment markedly increased the rate of tumor incidence but had no effect on tumor growth rate as measured by tumor doubling time. A comparison of tumor labeling index and mitotic index between the two groups showed no difference. A histological comparison of the mammary glands in the two groups showed no significant difference in number of hyperplastic alveolar nodules. However, ductal-alveolar development was more pronounced in the 0.15 M NaCl-treated control group; consistent with this observation was the finding of lower levels of serum prolactin in the T3-treated groups. Since mammary tumorigenesis was increased in T3-treated mice in spite of lower levels of serum prolactin, a well-known promoter of spontaneous mammary tumors in mice, a direct role for T3 in the process of tumor formation is suggested.

Nuclear thyroid hormone receptors in C3H/HeN mouse mammary glands and spontaneous tumors.

Saturable, high-affinity binding sites for 3,5,3'-triiodo-L-thyronine (T3) were identified in isolated nuclei and solubilized chromatin extracts of mammary glands, spontaneous mammary tumors, and liver from C3H/HeN mice. Receptor concentration in whole mammary gland nuclei (254 fmol/mg DNA) was only about one-half that of mouse liver nuclei (536 fmol/mg DNA), but in molecular weight (55,000) and in their affinity for various thyroid hormone analogues, the binding was essentially identical. Saturation analysis of T3 binding in a series of individual spontaneous mammary tumors and pooled lactating mammary glands indicated that the concentrations of T3-binding sites of the mammary gland are conserved in the transition to neoplasms and are somewhat increased in the largest tumors. Thyroxine binding was identical in capacity to T3 binding in mammary gland nuclei and nuclear extract but showed a higher binding capacity than did T3 in the largest tumors. High-performance molecular exclusion chromatography did show a difference between mammary gland and liver in the distribution of competible [125I]T3 binding between two macromolecular forms; the excluded peak (Mr greater than 450,000) comprised 56% of the T3 binding in the liver but only 9% in the mammary gland with the included peak (Mr 55,000) contributing the balance of binding in each case. Spontaneous mammary tumor resembled the mammary gland in the macromolecular distribution of specific T3 binding (16% excluded). Thymidine uptake showed only a modest decrease in the larger tumors (greater than 2.0 g), while nuclear histone acetylase activity was significantly decreased in this group. Neither measurement showed a significant correlation with T3 or thyroxine binding capacity.

Characterization of intestinal alkaline phosphatase expression and the tumorigenic potential of gamma-irradiated HeLa x fibroblast cell hybrids.

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in genetically stable hybrids which are nontumorigenic and no longer express the HeLa tumor-associated antigen, intestinal alkaline phosphatase (IAP). Previous analysis of spontaneous segregants of the nontumorigenic hybrid have implicated the loss of one copy of human fibroblast chromosome 11 with reexpression of IAP and tumorigenicity. This observation suggests that a putative HeLa tumor suppressor gene(s) is located on chromosome 11 and that this gene may be a negative regulator of the IAP gene. We have isolated several gamma-ray-induced mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 that were specifically selected for reexpression of IAP to further investigate the potential linkage between IAP regulation and the putative tumor suppressor locus. The GIMs have a wide range of cell morphology and level of IAP expression (nearly a factor of 40). The tumorigenicity of the GIMs was examined by s.c. injection into nude mice and all were found to be tumorigenic. The tumor volume-doubling time is in the range of 4 to 8 days for all the cell lines; however, the lag time to reach 500 mm3 tumor volume was significantly longer when the GIM IAP activity was low (less than 20% relative activity), suggesting perhaps that there is a threshold level of IAP expression required for tumor formation and selection for high IAP expression in vivo. However, studies with tumor reconstitutes of the GIMs and transfection studies with an IAP complementary DNA expression vector indicate that high IAP expression alone is not sufficient to confer rapid tumor growth. Therefore, while the data lend strong support to the continued tight correlation between IAP reexpression and tumorigenicity and to our proposal that the tumor suppressor may negatively regulate the IAP gene, it suggests that selection for other gene activities may be responsible for aggressive tumor growth in this cell hybrid system.

Meeting the UK driving vision standards with reduced contrast sensitivity.

PurposeThe visual standard to hold a UK driver's license since 2012 includes visual acuity (VA) measured indoors and the ability to read a car numberplate outdoors. Individuals with reduced contrast sensitivity may have greater visual difficulties outdoors. The agreement between the two tests in the presence of combined reduction in contrast sensitivity and VA was investigated.MethodsSimulation glasses ('sim-specs') were used to reduce both high-contrast VA and contrast sensitivity (CS). Following evaluation of the influence of sim-specs on VA and CS, levels 2 to 4 were chosen to give a range of VAs on either side of the driving standard of 6/12. Sixty-two participants wearing sim-specs then had VA tested with Snellen and ETDRS charts indoors, and ability to read a numberplate assessed outdoors as per DVLA regulations.ResultsSim-specs reduced VA and CS by ~0.10 logMAR VA per 0.10 logCS. The sensitivity of test chart VA <6/12 to correctly predict failure on the numberplate was 61% for Snellen and 56% for ETDRS.ConclusionFalse-negative and -positive rates were higher than in a previous study with uncorrected refractive error only. Reduced CS increased the lack of agreement between the two driving vision standards, which likely occurs as the VA test is performed indoors and the numberplate test outdoors. The increased likelihood of failing the numberplate test even though VA is 6/12 or better needs to be considered when advising patients on fitness to drive who have ocular disease such as cataract.

Murine A-myb: evidence for differential splicing and tissue-specific expression.

The myb gene family consists of three members, A-myb, B-myb and c-myb. The proteins encoded by these genes bind DNA in a sequence-specific manner and regulate transcription of target genes. In this communication, we report the nucleotide sequence of murine A-myb cDNA. This gene encodes a protein of 751 amino acids with an estimated molecular weight of 83 kDa. cDNA sequence analysis of multiple independent cDNA clones reveals the presence of alternatively spliced mRNAs that encode smaller proteins. Northern blot analysis of adult mouse tissue RNAs reveals A-myb expression predominantly in the testis, with very low levels of expression in the ovaries, spleen and brain. In situ hybridization analysis of adult mouse testis shows that this gene is expressed at high levels in type A spermatogonia (stem cells), and preleptotene and pachytene spermatocytes, with concomitant down-regulation of expression upon terminal differentiation of these cells into mature spermatozoa. This pattern of expression suggests that A-myb is involved in the control of proliferation and differentiation of spermatogonia. This potential function of A-myb in spermatogenesis is reminiscent of the role of c-myb in hematopoiesis.

Temporal patterns of A-myb and B-myb gene expression during testis development.

We recently reported the cloning and sequencing of the mouse A-myb proto-oncogene cDNA and the abundant expression of this mRNA primarily in the testis of adult mice. The A-myb mRNA is detectable by in situ hybridization specifically in the spermatogenic cells, and is downregulated during terminal differentiation. A low level of expression is observed in a few other tissues, including ovary, spleen and brain. We have extended those studies by examining A-myb and B-myb expression during testis development in the mouse. The A-myb and B-myb genes are both expressed in a cell- and stage-specific manner during testis development. The B-myb mRNA is expressed most highly in gonocytes of the fetal testis and in spermatogonia and early spermatocytes in the adult. B-myb expression decreases at day 18 post partum, coincident with the initial appearance of late pachytene spermatocytes. B-myb expression was also detectable in some interstitial cells of the fetal and adult testis. The A-myb mRNA was not detectable by in situ hybridization in fetal day 15.5 gonocytes but was detectable at a low abundance by RT-PCR in fetal and newborn mice. A-myb mRNA expression increased at post-natal day 10, when primary spermatocytes first appear. In the adult, the A-myb mRNA was expressed highly in a sub-population of spermatogonia and in primary spermatocytes, but was not detectable in spermatids. This expression of A-myb is consistent with the meiotic arrest that is observed in A-myb-deficient male mice. We conclude that B-myb may play a critical role in controlling the proliferation or differentiation of gonocytes and spermatogonia and possibly the somatic lineages as well, whereas A-myb is required for progression through the first meiotic prophase. These distinct roles for B-myb and A-myb during spermatogenesis may reflect distinct transactivation potentials of the two proteins. Further studies to determine the functions of A-myb and B-myb in the developing testis should improve our understanding of the molecular events associated with spermatogenesis and differentiation of the Sertoli and other somatic cell types of the testis.

Inhibition of growth of estrogen receptor positive and estrogen receptor negative breast cancer cells in culture by AA-etherA, a stable 2-5A derivative.

The design, chemical synthesis and biological activities of a nuclease-resistant, nontoxic bioactive 2-5A derivative, AA-etherA [i.e., adenylyl-(2'-5')-adenylyl-(2'-2")-9-[(2'-hydroxyethoxy)-methyl]adenine], are described as a new approach to the inhibition of breast cancer cell growth. AA-etherA inhibits DNA replication and cell division of both estrogen receptor positive (MCF-7) and estrogen receptor negative (BT-20) breast cancer cells in culture in a dose-dependent manner. Maximal inhibition in MCF-7 and BT-20 cells was obtained with 100 microM AA-etherA after four days of treatment, with an GI50 of 58 and 37 microM, respectively. AA-etherA is stable in the cytoplasm. Treated cells accumulate within the late G1/early S phase of the cell cycle and then progress only very slowly through S phase. AA-etherA does not activate RNase L, as do 2-5A and other 2-5A derivatives, nor does it increase p68 kinase (PKR) content of the cells. High resolution, two-dimensional protein gel electrophoresis reveals twofold or greater inhibition of synthesis of 92 proteins out of 682 proteins that were reproducibly detected as high quality spots with average rates of synthesis of > or = 20 p.p.m. in untreated cells. The specificity of the effects of AA-etherA on select proteins and its failure to activate RNase L indicate that AA-etherA does not act through a general effect on mRNA translation or stability, but rather inhibits cell proliferation through a block to DNA replication, with a concommitant reduction in the synthesis of specific proteins, some of which may be required for cell cycle transit. Two likely targets to account for the AA-etherA inhibition of DNA replication are DNA topoisomerase I, which is inhibited by AA-etherA in other cell lines, and thymidine kinase, which could be inhibited in a manner similar to the effect of acyclovir. These data indicate that 2-5A analogs, particularly bifunctional 2-5A analogs like AA-etherA, will be useful for controlling cancer cell growth. Further development of such 2-5A analogs may provide highly specific compounds for chemotherapy and chemoprevention.

T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer.

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.

Interaction of amiodarone and desethylamiodarone with solubilized nuclear thyroid hormone receptors.

The mechanisms of action of the potent antiarrhythmic drug amiodarone are unknown. However, amiodarone and its abundant metabolite, desethylamiodarone, bear a striking structural resemblance to thyroid hormones. In addition, certain cardiac electrophysiologic effects of amiodarone treatment are similar to those of hypothyroidism. These facts suggest that amiodarone or desethylamiodarone could be acting, in part, by blocking thyroid hormone action. Because thyroid hormones are known to act through nuclear receptor proteins, the binding of amiodarone and desethylamiodarone was measured to nuclear extracts derived from human lymphocytes, bovine atrium and ventricle and rat liver. The capacity of increasing concentrations of amiodarone and desethylamiodarone nuclear extracts to block receptor binding of radiolabeled triiodothyronine (T3) in a standard in vitro competition assay was tested. Nuclear extracts demonstrated only minimal binding to amiodarone. However, all receptor preparations had substantial affinities (KD) for the desethyl analog: lymphocyte, 8.6 microM; atrium, 35.0 microM; ventricle, 26.9 microM and liver, 8.6 microM. Desethylamiodarone accumulates in very large quantities in parenchymatous organs during long-term amiodarone treatment. Taking its usual therapeutic serum level (about 4 microM or 2.7 micrograms/ml) as an estimate of intranuclear concentration, desethylamiodarone would partially saturate nuclear thyroid hormone receptors in several different tissues, including the heart. Thus, amiodarone treatment may exert some of its electrophysiologic effects by metabolic conversion to desethylamiodarone. This metabolite may then exclude thyroid hormone from nuclear receptor sites within the myocardium.

Mechanisms and control of embryonic genome activation in mammalian embryos.

Activation of transcription within the embryonic genome (EGA) after fertilization is a complex process requiring a carefully coordinated series of nuclear and cytoplasmic events, which collectively ensure that the two parental genomes can be faithfully reprogrammed and restructured before transcription occurs. Available data indicate that inappropriate transcription of some genes during the period of nuclear reprogramming can have long-term detrimental effects on the embryo. Therefore, precise control over the time of EGA is essential for normal embryogenesis. In most mammals, genome activation occurs in a stepwise manner. In the mouse, for example, some transcription occurs during the second half of the one-cell stage, and then a much greater phase of genome activation occurs in two waves during the two-cell stage, with the second wave producing the largest onset of de novo gene expression. Changes in nuclear structure, chromatin structure, and cytoplasmic macromolecular content appear to regulate these periods of transcriptional activation. A model is presented in which a combination of cell cycle-dependent events and both translational and posttranslational regulatory mechanisms within the cytoplasm play key roles in mediating and regulating EGA.

Mechanistic and developmental aspects of genetic imprinting in mammals.

Genetic imprinting in mammals allows the recognition and differential expression of maternal and paternal alleles of certain genes. Recent results from a number of laboratories indicate that, at least for some genes, gametic imprints, which must exist in order to mark chromosomes or genes as having been transmitted via sperm or ovum, are not by themselves sufficient to determine allele expression. Other postfertilization events are required, and these events are subject to both tissue-specific and developmental stage-specific regulation. Changes in imprinted gene methylation during preimplantation and fetal life indicate that the establishment of additional allele-specific modifications is likely to contribute to imprinted regulation. Disruptions in imprinting processes, loss of imprints, and loss of nonimprinted alleles through uniparental disomy are likely to contribute to a variety of developmental abnormalities and pathological conditions in both mice and humans.