Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.     

Phylogenetic footprinting of hypersensitive site 3 of the beta-globin locus control region

Hypersensitive site 3 (HS3) of the beta-like globin locus control region has been implicated as an important regulator of the beta-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two gamma genes (galago gamma is embryonic; human gamma is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of gamma in adult life).     

The human S mu bp-2, a DNA-binding protein specific to the single-stranded guanine-rich sequence related to the immunoglobulin mu chain switch region

We have cloned the cDNA encoding the human homologue of S mu bp-2, which binds to single-stranded DNA with 5'-phosphorylated guanine-rich sequences related to the immunoglobulin mu chain switch (S mu) region. The deduced amino acid sequences of the mouse and human S mu bp-2 are 76.5% homologous and contain motifs conserved among helicases. We have identified a domain essential for DNA binding at residues 638-786. The binding domain is less conserved (63% homologous) than the putative catalytic domain of N-terminal half containing most of the helicase motifs (85% homologous). The human and mouse S mu bp-2 have similar, although slightly different, binding specificities. Although the mouse S mu bp-2 preferentially binds to the mouse S mu motif (GGGGT), the human S mu bp-2 binds equally well to the human (GGGCT) and mouse S mu motifs. The human S mu bp-2 gene was mapped to chromosome 11 q13.2-q13.4 by in situ hybridization.     

Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.     

Identification and purification of a spinach chloroplast DNA-binding protein that interacts specifically with the plastid psaA-psaB-rps14 promoter region

OBJECTIVE: To catalog a series of rare lesions of the posterior fossa that appeared with unusual initial retrocochlear symptoms and signs and to make the reader more aware of these unusual lesions with a view to improving initial assessment and treatment planning. STUDY DESIGN: The study was a retrospective case review of seven patients. SETTING: Multidisciplinary team evaluation in a tertiary hospital referral center. PATIENTS: Patients with unusual lesions of the cerebellopontine angle and posterior fossa with initial retrocochlear symptoms and signs were included. INTERVENTIONS: Diagnostic and therapeutic. MAIN OUTCOME MEASURES: Hearing preservation and balance function. RESULTS: The rare lesions presented include two aneurysms of the anterior inferior cerebellar artery, one giant basilar artery aneurysm, and one each of the following neoplasms: endodermal cyst, choroid plexus papilloma, cavernous angioma, and ependymoma. CONCLUSIONS: A close working relationship among the otolaryngologist, neurotologist, neurosurgeon, and neuroradiologist is necessary to accurately evaluate these unusual cerebellopontine angle lesions and effect the best treatment outcome.     

A novel nucleic acid-binding protein that interacts with human rad51 recombinase

Using the yeast two-hybrid system, we isolated a cDNA encoding a novel human protein, named Pir51, that strongly interacts with human Rad51 recombinase. Analysis in vitro confirmed the interaction between Rad51 and Pir51. Pir51 mRNA is expressed in a number of human organs, most notably in testis, thymus, colon and small intestine. The Pir51 gene locus was mapped to chromosome 12p13.1-13. 2 by fluorescence in situ hybridization. The Pir51 protein was expressed in Escherichia coli and purified to near homogeneity. Biochemical analysis shows that the Pir51 protein binds both single- and double-stranded DNA, and is capable of aggregating DNA. The protein also binds RNA. The Pir51 protein may represent a new member of the multiprotein complexes postulated to carry out homologous recombination and DNA repair in mammalian cells.     

A novel RING finger protein, Vps8p, functionally interacts with the small GTPase, Vps21p, to facilitate soluble vacuolar protein localization

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete soluble vacuolar hydrolases. Here we report the characterization of the gene product affected in one of these mutants, Vps8p. Polyclonal antiserum raised against a trpE-Vps8 fusion protein specifically detects a 134-kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that Vps8p is distributed between a low speed membrane pellet fraction and a high speed membrane pellet fraction. The lack of a hydrophobic domain in Vps8p suggests that Vps8p peripherally associates with a membrane(s). This association was found to depend on the function of Vps21p, a member of the Rab/Ypt/Sec4 family of small GTPases. In vps21 null mutant cells, Vps8p is found in the cytosol. In addition, overexpression of Vps21p partially suppresses a vps8 null mutant, indicating that Vps8p and Vps21p functionally interact. Vps8p contains a C-terminal cysteine-rich region that conforms to the H2 variant of the RING finger Zn2+ binding motif. Truncation of this C-terminal region partially compromises Vps8p function. While vps8 null mutant strains missort and secrete soluble vacuolar hydrolases, the integral vacuolar membrane protein, alkaline phosphatase (ALP), is sorted to the vacuole and matured normally. In addition, when vps8 mutants are combined with endocytic or late secretory pathway mutants (end3 or sec1, respectively), ALP is still delivered to the vacuole. These observations indicate that ALP is sorted to the vacuole in a Vps8p-independent manner, possibly via an alternative vesicle carrier.     

Genetic dissection of a mammalian replicator in the human beta-globin locus

The timing and localization of DNA replication initiation in mammalian cells are heritable traits, but it is not known whether initiation requires specific DNA sequences. A site-specific recombination strategy was used to show that DNA sequences previously identified as replication initiation sites could initiate replication when transferred to new chromosomal locations. An 8-kilobase DNA sequence encompassing the origin of DNA replication in the human beta-globin locus initiated replication in the simian genome. Specific deletions within the globin origin did not initiate replication in these chromosomal sites. These data suggest that initiation of DNA replication in mammalian cells requires specific sequence information and extend the replicon hypothesis to higher eukaryotes.     

Conserved elements containing NF-E2 and tandem GATA binding sites are required for erythroid-specific chromatin structure reorganization within the human beta-globin locus control region

Proper expression of the genes of the human beta-globin gene locus requires the associated locus control region (LCR). Structurally, the LCR is defined by the presence of four domains of erythroid-specific chromatin structure. These domains, which have been characterized as DNase I hypersensitive sites (HSs), comprise the active elements of the LCR. The major focus of this research is to define the cis -acting elements which are required for the formation of these domains of unique chromatin structure. Our previous investigations on the formation of LCR HS4 demonstrated that NF-E2 and tandem, inverted GATA binding sites are required for the formation of the native HS. Similarly arranged NF-E2 and tandem GATA sites are present within the core regions of the other human LCR HSs and are evolutionarily conserved. Using site-directed mutagenesis of human HSs 2 and 3 we have tested the hypothesis that these NF-E2 and GATA sites are common requirements for the formation of all LCR HSs. We find that mutation of these elements, and particularly the GATA elements, results in a decrease or complete loss of DNase I hypersensitivity. These data imply the presence of common structural elements within the core of each LCR HS which are required for erythroid-specific chromatin structure reorganization.     

Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.     

A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A

Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast two-hybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single- or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair.