A novel imprinted gene, encoding a RING zinc-finger protein, and overlapping antisense transcript in the Prader-Willi syndrome critical region.

We describe a complex imprinted locus in chromosome 15q11-q13 that encodes two genes, ZNF127 and ZNF127AS. The ZNF127 gene encodes a protein with a RING (C3HC4) zinc-finger and multiple C3H zinc-finger motifs, the former being closely related to a protein from variola major virus, the smallpox etiological agent. These motifs allow prediction of ZNF127 function as a ribonucleoprotein. The intronless ZNF127 gene is expressed ubiquitously, but the entire coding sequence and 5' CpG island overlaps a second gene, ZNF127AS, that is transcribed from the antisense strand with a different transcript size and pattern of expression. Allele-specific analysis shows that ZNF127 is expressed only from the paternal allele. Consistent with this expression pattern, in the brain the ZNF127 5' CpG island is completely unmethylated on the paternal allele but methylated on the maternal allele. Analyses of adult testis, sperm and fetal oocytes demonstrates a gametic methylation imprint with unmethylated paternal germ cells. Recent findings indicate that ZNF127 is part of the coordinately regulated imprinted domain affected in Prader-Willi syndrome patients with imprinting mutations. Therefore, ZNF127 and ZNF127AS are novel imprinted genes that may be associated with some of the clinical features of the polygenic Prader-Willi syndrome.

Imprinting of a RING zinc-finger encoding gene in the mouse chromosome region homologous to the Prader-Willi syndrome genetic region.

A novel locus in the human Prader-Willi syndrome (PWS) region encodes the imprinted ZNF127 and antisense ZNF127AS genes. Here, we show that the mouse ZNF127 ortholog, Zfp127, encodes a homologous putative zinc-finger polypeptide, with a RING (C3HC4) and three C3H zinc-finger domains that suggest function as a ribonucleoprotein. By the use of RT-PCR across an in-frame hexamer tandem repeat and RNA from a Mus musculus x M.spretus F1interspecific cross, we show that Zfp127 is expressed only from the paternal allele in brain, heart and kidney. Similarly, Zfp127 is expressed in differentiated cells derived from androgenetic embryonic stem cells and normal embryos but not those from parthogenetic embryonic stem cells. We hypothesize that the gametic imprint may be set, at least in part, by the transcriptional activity of Zfp127 in pre- and post-meiotic male germ cells. Therefore, Zfp127 is a novel imprinted gene that may play a role in the imprinted phenotype of mouse models of PWS.

Gene structure, DNA methylation, and imprinted expression of the human SNRPN gene.

The human SNRPN (small nuclear ribonucleoprotein polypeptide N) gene is one of a gene family that encode proteins involved in pre-mRNA splicing and maps to the smallest deletion region involved in the Prader-Willi syndrome (PWS) within chromosome 15q11-q13. Paternal only expression of SNRPN has previously been demonstrated by use of cell lines from PWS patients (maternal allele only) and Angelman syndrome (AS) patients (paternal allele only). We have characterized two previously unidentified 5' exons of the SNRPN gene and demonstrate that exons -1 and 0 are included in the full-length transcript. This gene is expressed in a wide range of somatic tissues and at high, approximately equal levels in all regions of the brain. Both the first exon of SNRPN (exon -1) and the putative transcription start site are embedded within a CpG island. This CpG island is extensively methylated on the repressed maternal allele and is unmethylated on the expressed paternal allele, in a wide range of fetal and adult somatic cells. This provides a quick and highly reliable diagnostic assay for PWS and AS, which is based on DNA-methylation analysis that has been tested on > 100 patients in a variety of tissues. Conversely, several CpG sites approximately 22 kb downstream of the transcription start site in intron 5 are preferentially methylated on the expressed paternal allele in somatic tissues and male germ cells, whereas these same sites are unmethylated in fetal oocytes. These findings are consistent with a key role for DNA methylation in the imprinted inheritance and subsequent gene expression of the human SNRPN gene.

Organization and sequence of the human P gene and identification of a new family of transport proteins.

We have determined the structure, nucleotide sequence, and polymorphisms of the human P gene. Mutations of the P gene result in type II oculocutaneous albinism (OCA2) in humans and pink-eyed dilution (p) in mice. We find that the human P gene is quite large, consisting of 25 exons spanning 250 to 600 kb in chromosome segment 15q11-q13. The P polypeptide appears to define a novel family of small molecule transporters and may be involved in transport of tyrosine, the precursor to melanin synthesis, within the melanocyte. These results provide the basis for analyses of patients with OCA2 and may point toward eventual pharmacologic treatment of this and related disorders of pigmentation.

A sequence in the rat Pit-1 gene promoter confers synergistic activation by glucocorticoids and protein kinase-C.

The 5'-flanking region of the gene for Pit-1, a pituitary-specific transcription factor, was isolated from a rat liver genomic library and sequenced. Expression of a reporter construct containing Pit-1 promoter sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was assessed by transient transfection in rat pituitary GH4C1 cells. Treatment of transfected cells with either dexamethasone (DEX) for 48 h or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) for the final 20 h of the 48-h posttransfection period had minimal effects on CAT expression. However, CAT activity was elevated about 20-fold when transfected cells were treated with both DEX and TPA. This apparent synergistic activation was lost when DEX treatment was also limited to the final 20 h of the 48-h posttransfection period, suggesting that a time-dependent accumulation of a DEX-induced gene product might be involved. This putative DEX-induced product appeared to be relatively stable, because synergistic activation was observed in cells treated with DEX alone for 36 h, followed by a 10-h incubation without DEX before the addition of TPA. The Pit-1 gene promoter region between -210 and -142 from the transcription start site conferred synergistic regulation by DEX and TPA when placed upstream of position -105 in the herpes viral thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional imprinting and epigenetic modification of the human SNRPN gene.

The SNRPN gene encodes a small nuclear ribonucleoprotein subunit, SmN, thought to be involved in splicing of pre-mRNA. A closely related protein, SmB/B', is constitutively expressed in all tissues except the brain, where SmN is predominantly expressed. The mouse homolog of the SNRPN gene has been shown to be functionally imprinted in mouse brain, being expressed only from the paternally derived chromosome. SNRPN has been mapped to human chromosome 15q11-q13 within the shortest region of deletion overlap for the Prader-Willi syndrome. We have now demonstrated functional imprinting of the human SNRPN gene using reverse transcription followed by the polymerase chain reaction (RT-PCR). No expression was observed in cultured skin fibroblasts of Prader-Willi patients, but was found in all Angelman patients and normal controls examined. We have also demonstrated a parent-specific DNA methylation imprint within intron 5 of the SNRPN gene, which suggests an epigenetic mechanism by which parent-specific expression of this gene might be inherited. Our findings indicate that SNRPN is expressed only from the paternally derived chromosome 15 in humans and therefore may fulfill one major criterion for being involved in the pathogenesis of the Prader-Willi syndrome.

A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albinism.

The mouse pink-eyed dilution (p) locus on chromosome 7 is associated with defects of skin, eye and coat pigmentation. Mutations at p cause a reduction of eumelanin (black-brown) pigment and altered morphology of black pigment granules (eumelanosomes), but have little effect on pheomelanin (yellow-red) pigment. We show here that the human complementary DNA DN10, linked to the p locus in mice, identifies the human homologue (P) of the mouse p gene, and appears to encode an integral membrane transporter protein. The expression pattern of this gene in various p mutant mice correlates with the pigmentation phenotype; moreover, an abnormally sized messenger RNA is detected in one mutant, p(un), which reverts to the normal size in p(un) revertants. The human P gene corresponds to the D15S12 locus within the chromosome segment 15q11-q13, which is typically deleted in patients with Prader-Willi and Angelman syndrome (see ref. 5 for review). These disorders are phenotypically distinct, depending on the parent of origin of the deleted chromosome, but both syndromes are often associated with hypopigmentation of the skin, hair and eyes (see ref. 8 for review), and deletion of the P gene may be responsible for this hypopigmentation. In addition, we report a mutation in both copies of the human P gene in one case of tyrosinase-positive (type II) oculocutaneous albinism, recently linked to 15q11-q13 (ref. 9).

The homeodomain protein, Pit-1/GHF-1, is capable of binding to and activating cell-specific elements of both the growth hormone and prolactin gene promoters.

Studies were conducted to determine whether the trans-acting protein Pit-1/GHF-1 can bind to and activate promoter elements in both the GH and PRL genes that are necessary for cell-specific expression. Four pituitary cell lines that differentially express the endogenous GH and PRL genes were examined for their ability to activate GH and PRL promoter constructs containing sequences necessary for cell-specific expression (CSEs). Plasmids containing one CSE, -96 PRL and -104 GH, were similarly expressed in each of the four cell lines. Of the plasmids containing two CSEs, -173 PRL was always activated to a greater extent than -145 GH, with this relative activation being stronger in GC and GH1 cells than in 235-1 and GH4C1 cells. Protein-DNA binding assays were used to show that the GH and PRL CSEs specifically bound two highly abundant nuclear proteins (31 and 33 kDa). The two proteins were present at similar levels in all four pituitary cell lines and were recognized by a Pit-1/GHF-1 antibody. In contrast, HeLa and Rat2 cells did not activate transfected GH or PRL plasmids and did not contain nuclear proteins that specifically bound to the GH and PRL CSEs. However, cotransfection of these cells with the expression vector RSV-Pit-1/GHF-1 resulted in the activation of -173 PRL and -145 GH (PRL greater than GH). HeLa cells transfected with RSV-Pit-1/GHF-1 also contained 31- and 33-kDa nuclear proteins that bound to the GH and PRL CSEs. These results show that Pit-1/GHF-1 is present at levels in pituitary cell lines that are sufficient to activate the minimal elements in both the GH and PRL promoters necessary for cell-specific expression of these genes.

CR3 (CD11b/CD18) expresses one binding site for Arg-Gly-Asp-containing peptides and a second site for bacterial lipopolysaccharide.

Polymorphonuclear leukocytes (PMN) from three patients deficient in the CD18 family of receptors (LFA-1, CR3, and p150,95) exhibited an inability to bind erythrocytes coated with C3bi or bacterial LPS. These observations confirm that the CD18 family, and CR3 in particular, can bind the structurally dissimilar molecules C3bi and LPS. Further studies showed that LPS and C3bi bind to CR3 at distinct sites. mAb OKM10 against CR3 blocked binding of C3bi to PMN but did not block the binding of LPS. In contrast, mAb 904, directed against a different epitope on CR3, blocked binding of LPS to PMN but not binding of C3bi, thus suggesting that different regions of CR3 were involved in binding these two ligands. In addition, synthetic peptides based on the sequence in C3bi recognized by CR3 competitively blocked the binding of C3bi to CR3 but did not block the binding of LPS. Rather, occupation of the peptide binding site on CR3 by the synthetic peptides enhanced binding of LPS. These results indicate that CR3 has two distinct binding sites, one that recognizes ligands composed of protein and a second that recognizes LPS.

C3bi receptor (complement receptor type 3) recognizes a region of complement protein C3 containing the sequence Arg-Gly-Asp.

Human phagocytes express a receptor termed complement receptor type 3 (CR3) that recognizes the complement protein fragment C3bi. We show here that CR3 recognizes a region of C3 that contains the sequence Arg-Gly-Asp (RGD). CR3 is down-modulated upon spreading of macrophages on surfaces coated with a synthetic 21-residue peptide from C3 (residues 1383-1403). This peptide was also attached to erythrocytes by coupling myristic acid to its amino terminus and allowing the myristoylated peptide to bind to erythrocytes through hydrophobic interactions. Erythrocytes coated with this RGD-containing segment of C3 were bound by macrophages, and binding could be blocked by specific monoclonal antibodies against CR3. Since CR3 recognizes a peptide sequence that contains the RGD triplet, it appears to be a member of a larger family of adhesion-promoting receptors that recognize RGD-containing proteins. However, since CR3 does not recognize a hexapeptide containing RGD, we presume that residues beyond the RGD triplet contribute to binding. We have compared the RGD-containing region of fibronectin and vitronectin, proteins known to be recognized by means of their RGD-containing regions, with those in human and murine C3. A striking homology is observed over an approximately equal to 50 amino acid sequence present in all four proteins. We suggest that this extended region of homology contains a structure recognized by adhesion-promoting receptors.

Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide.

We report here that human macrophages bind Escherichia coli by recognizing bacterial lipopolysaccharide (LPS). Purified LPS was inserted into erythrocyte membranes, and the resulting LPS-coated red cells were bound by macrophages with the same temperature and cation dependence as observed for E. coli. When receptors for LPS were withdrawn from the plasma membrane by spreading the macrophages on LPS-coated surfaces, the binding of E. coli was blocked. We have also identified the receptors on macrophages that recognize LPS. Macrophages express three structurally homologous cell surface proteins, CR3, lymphocyte function-associated antigen (LFA-1), and p150,95. We used surface-bound monoclonal antireceptor antibodies to selectively remove these proteins from the apical surface of macrophages. We found that each of these proteins mediated the binding of E. coli to macrophages.

Interferon-gamma depresses binding of ligand by C3b and C3bi receptors on cultured human monocytes, an effect reversed by fibronectin.

Cultivation of human monocytes with recombinant IFN-gamma causes a 5-10-fold depression in their binding of EC3b or EC3bi. This effect is observed within 18 h and is expressed for 5 d in the presence of 100 U/ml IFN-gamma. The capacity of IFN-gamma-treated phagocytes to bind EC3b and EC3bi is fully restored if the phagocytes are allowed to spread for 45 min on surfaces coated with Fn. IFN-gamma-treated cells express normal levels of cell surface C3b and C3bi receptors as measured with monoclonal anti-receptor antibodies, and spreading on Fn does not alter receptor number. We conclude that cultivation with IFN-gamma causes a change in the nature of these receptors that prevents them from interacting with ligand. Immunoelectron microscopy shows that C3bi receptors are expressed on the apical surface of the IFN-gamma-treated MO and that these receptors exhibit normal capacity to migrate in the plane of the membrane. Thus, the nature of the change caused by IFN-gamma is not related to changes in receptor number, location, or mobility. While spreading of IFN-gamma-treated cells on Fn enables C3 receptors to bind ligand, it does not enable them to promote phagocytosis. Treatment of cells with PMA alone does not affect binding or phagocytosis, but treatment of cells with both Fn and PMA enables cells to phagocytose EC3b and EC3bi. These data indicate that the binding and signaling activities of C3 receptors are separately regulated. Fn enables receptors to bind ligand and PMA enables them to signal phagocytosis.

Shoulder function following early mobilization of the shoulder after mastectomy and axillary dissection.

A prospective clinical trial was conducted to determine the effect of axillary node dissection for breast carcinoma on shoulder function and seroma production. 59 Operations were carried out in 57 patients. The patients were divided into two groups. In group A, shoulder exercises were started, under the guidance of a physiotherapist, immediately following surgery, and in group B the exercises were begun on the seventh postoperative day. A full range of motion within six months was achieved in 25 patients of group A (81%) and in 22 of group B (79%). There was no significant difference in wound drainage between the two groups. Restricted shoulder movement was often seen after local wound complications following axillary radiotherapy or after seriously disturbed wound healing.