Effect of intravenous coadministration of human stroma cell lines on engraftment of long-term repopulating clonal myelodysplastic syndrome cells in immunodeficient mice.

Engraftment of clonal hematopoietic precursor cells from patients with myelodysplastic syndrome (MDS) in immunodeficient mice has been difficult to achieve by intravenous (i.v.) injection. We used i.v. coadministration of the human marrow stroma cell line HS27a with CD34+ MDS cells in Nod.cg-Prkdc(scid) Il2rg(tm1wjll) (NSG) mice to provide signals that would facilitate engraftment. Hematopoietic cells from 24 MDS patients were transplanted. Cells from all patients were engrafted, and engraftment was documented in 44 of 46 evaluable mice (95%). Immunohistochemistry revealed human HS27a stroma colocalizing with human hematopoietic cells in mouse spleens. Human CD34+ precursors harvested from marrow and spleen of primary murine recipients, when combined with HS27a cells, were also engrafted successfully in secondary NSG recipients, showing persistence of the original clonal characteristics. This observation supports the concept that clonal markers were present in long-term repopulating cells. We suggest that HS27a stroma cells 'traveled' in direct contact with hematopoietic precursors and enabled their propagation. An essential signal for engraftment appears to be CD146, which is prominently expressed on HS27a cells. This xenotransplantation model will allow to further dissect signals that control engraftment of MDS cells and should be amenable to in vivo treatment studies.

Awareness of memory functioning in early Alzheimer's disease: lessons from a comparison with healthy older people and young adults.

To compare awareness of memory in people with early Alzheimer's disease (AD) with older and younger control groups in order to clarify the contributions of disease and ageing. Twenty-one individuals with early AD, 32 older people (OP) and 32 younger people (YP) estimated their performance before and following each of four tests of memory. Those with AD significantly overestimated performance prior to testing, confirming the presence of unawareness for prediction. Their estimates were adjusted for age in a similar way to those of OP, who predicted their performance accurately. Younger people significantly underestimated their performance. Following memory tests, YP and those with AD significantly adjusted their ratings towards greater accuracy but on average those with AD still overestimated. There were variations in post-test ratings from over to under prediction in all three groups. Heightened awareness post-performance may open possibilities of cognitive rehabilitation to consolidate momentary into more long-term awareness, whilst the operation of possible psychosocial influences suggests that emotion oriented interventions to increase acceptance and decrease shame might be helpful.

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.

Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation.

We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

Independent formation of DnaseI hypersensitive sites in the murine beta-globin locus control region.

Mammalian beta-globin loci are composed of multiple orthologous genes whose expression is erythroid specific and developmentally regulated. The expression of these genes both from the endogenous locus and from transgenes is strongly influenced by a linked 15-kilobase region of clustered DNaseI hypersensitive sites (HSs) known as the locus control region (LCR). The LCR encompasses 5 major HSs, each of which is highly homologous among humans, mice, and other mammals. To analyze the function of individual HSs in the endogenous murine beta-globin LCR, we have used homologous recombination in embryonic stem cells to produce 5 mouse lines, each of which is deficient for 1 of these major HSs. In this report, we demonstrate that deletion of the conserved region of 5'HS 1, 2, 3, 4, or 5/6 abolishes HS formation at the deletion site but has no influence on the formation of the remaining HSs in the LCR. Therefore, in the endogenous murine locus, there is no dominant or initiating site whose formation must precede the formation of the other HSs. This is consistent with the idea that HSs form autonomously. We discuss the implications of these findings for current models of beta-globin regulation.

Conservation of sequence and structure flanking the mouse and human beta-globin loci: the beta-globin genes are embedded within an array of odorant receptor genes.

In mouse and human, the beta-globin genes reside in a linear array that is associated with a positive regulatory element located 5' to the genes known as the locus control region (LCR). The sequences of the mouse and human beta-globin LCRs are homologous, indicating conservation of an essential function in beta-globin gene regulation. We have sequenced regions flanking the beta-globin locus in both mouse and human and found that homology associated with the LCR is more extensive than previously known, making up a conserved block of approximately 40 kb. In addition, we have identified DNaseI-hypersensitive sites within the newly sequenced regions in both mouse and human, and these structural features also are conserved. Finally, we have found that both mouse and human beta-globin loci are embedded within an array of odorant receptor genes that are expressed in olfactory epithelium, and we also identify an olfactory receptor gene located 3' of the beta-globin locus in chicken. The data demonstrate an evolutionarily conserved genomic organization for the beta-globin locus and suggest a possible role for the beta-globin LCR in control of expression of these odorant receptor genes and/or the presence of mechanisms to separate regulatory signals in different tissues.

Description and targeted deletion of 5' hypersensitive site 5 and 6 of the mouse beta-globin locus control region.

The most upstream hypersensitive site (HS) of the beta-globin locus control region (LCR) in humans (5' HS 5) and chickens (5' HS 4) can act as an insulating element in some gain of function assays and may demarcate a beta-globin domain. We have mapped the most upstream HSs of the mouse beta-globin LCR and sequenced this region. We find that mice have a region homologous to human 5' HS 5 that is associated with a minor HS. In addition we map a unique HS upstream of 5' HS 5 and refer to this novel site as mouse 5' HS 6. We have also generated mice containing a targeted deletion of the region containing 5' HS 5 and 6. We find that after excision of the selectable marker in vivo, deletion of 5' HS 5 and 6 has a minimal effect on transcription and does not prevent formation of the remaining LCR HSs. Taken together these findings suggest that the most upstream HSs of the mouse beta-globin LCR are not necessary for maintaining the beta-globin locus in an active configuration or to protect it from a surrounding repressive chromatin environment.

The beta-globin LCR is not necessary for an open chromatin structure or developmentally regulated transcription of the native mouse beta-globin locus.

The murine beta-globin locus control region (LCR) was deleted from its native chromosomal location. The approximately 25 kb deletion eliminates all sequences and structures homologous to those defined as the human LCR. In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal. Thus, in the native murine beta-globin locus, the LCR is necessary for normal levels of transcription, but other elements are sufficient to establish the open chromatin structure, transcription, and developmental specificity of the locus. These findings suggest a contributory rather than dominant function for the LCR in its native location.

The locus control region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells.

Studies in many systems have led to the model that the human beta-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the beta-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous beta-globin locus in an erythroid background, transcription of all beta-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the beta-globin locus are not affected by the deletion. In contrast, deletion of 5'HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on beta-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an "open" conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the beta-like globin genes.

Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus.

The beta-globin locus control region (LCR) is a complex regulatory element that is essential for the appropriate red cell-specific expression of all cis-linked beta-globin genes. Of the five hypersensitive sites that define the LCR, only 5'HS2 has been shown to augment gene expression in vitro in both transient and stable assays, as well as in transgenic mice. Thus, 5'HS2 has been assumed to be an important element for the function of the LCR in vivo. We have utilized homologous recombination in murine embryonic stem (ES) cells and phenotypic analysis in derived mice to investigate the function of 5'HS2 in its normal chromosomal position in the murine beta-globin locus. Replacement of 5'HS2 with a selectable marker gene (delta HS2 + neo) causes a 2-5-fold reduction in expression of all of the genes in the locus, and a more pronounced effect (10-12-fold) on the most 5' embryonic globin gene, Ey, when expression of this gene is first detectable during embryogenesis. The mutation produces no alterations in the developmental timing of expression of the globin genes. When homozygous, the deletion/replacement mutation is lethal in utero, with the embryos dying during the stage of yolk sac and early fetal liver erythropoiesis. To distinguish phenotypic effects resulting from the deletion of 5'HS2 from those attributable to insertion of the selectable marker, the selectable marker was removed by expressing the FLP site-specific recombinase in ES cells harboring the homologous recombination event. Mice derived from these ES cells (delta HS2 delta neo) demonstrated nearly full expression of all the beta-like globin genes on the mutated chromosome. These results indicate that although 5'HS2 demonstrates significant regulatory activities in a variety of assays, deletion of this element from the endogenous beta-globin locus has no significant effect on the timing or extent of expression of the locus. In addition, this result emphasizes that when using homologous recombination to analyze complex regulatory elements in vivo, the inserted selectable marker must be removed to avoid influencing the phenotype of the mutation.

Novel putative receptor tyrosine kinase encoded by the melanoma-inducing Tu locus in Xiphophorus.

Malignant melanoma in Xiphophorus fish hybrids is caused by the activity of a dominant oncogene Tu. By combining genetic and molecular approaches, we have isolated the melanoma oncogene. We show that its level of expression correlates with the degree of malignancy of the tumour. The corresponding proto-oncogene is developmentally regulated. The Tu gene codes for a novel receptor tyrosine kinase which is closely related to the receptor for epidermal growth factor.

Patterns of nuclear lamins in diverse animal and plant cells and in germ cells as revealed by immunofluorescence microscopy with polyclonal and monoclonal antibodies.

Polyclonal and monoclonal antibodies against rat liver nuclear lamins have been used to evaluate the immunological cross-reactivity of lamins with a given antibody in a variety of animal and plant cells. The results indicated that lamins of all vertebrate cells but not invertebrate cells share at least one antigenic determinant, resulting in immunological cross-reaction with polyclonal antisera to lamina from rat liver. The range of cross-reaction with monoclonal antibody to rat lamins includes all mammalian cells tested but we observed no reaction with other vertebrate and invertebrate cells. Thus, by means of immunological cross-reaction a less stringently conserved pattern is observed for lamins than, for example, cytoskeletal proteins. We have also investigated the fate of the nuclear lamins during meiosis in testes and ovaries of the mouse. Lamins are absent from male meiotic cells and during oogenesis in meiotic prophases.