Mutation analysis in 54 propionic acidemia patients.

Deficiency of propionyl CoA carboxylase (PCC), a dodecamer of alpha and beta subunits, causes inherited propionic acidemia. We have studied, at the molecular level, PCC in 54 patients from 48 families comprised of 96 independent alleles. These patients of various ethnic backgrounds came from research centers and hospitals in Germany, Austria and Switzerland. The thorough clinical characterization of these patients was described in the accompanying paper (Grünert et al. 2012). In all 54 patients, many of whom originated from consanguineous families, the entire PCCB gene was examined by genomic DNA sequencing and in 39 individuals the PCCA gene was also studied. In three patients we found mutations in both PCC genes. In addition, in many patients RT-PCR analysis of lymphoblast RNA, lymphoblast enzyme assays, and expression of new mutations in E.coli were carried out. Eight new and eight previously detected mutations were identified in the PCCA gene while 15 new and 13 previously detected mutations were found in the PCCB gene. One missense mutation, p.V288I in the PCCB gene, when expressed in E.coli, yielded 134% of control activity and was consequently classified as a polymorphism in the coding region. Numerous new intronic polymorphisms in both PCC genes were identified. This study adds a considerable amount of new molecular data to the studies of this disease.

Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling.

Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shi(ts), mutant nerve terminals. Hypomorphic awd alleles display shi(ts)-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. We suggest a model in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo.

Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns.

Stably transfected cell lines containing the normal human growth hormone (hGH-N) and human growth hormone-variant (hGH-V) genes have been established in order to study the expression of these two highly homologous genes. Each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse C127 cells. The transfected cells containing the hGH-N gene secrete two hGH proteins, 91% migrating at 22 kD and 9% migrating at 20 kD, the same relative proportions synthesized in vivo by the human pituitary. S1 nuclease analysis of mRNA from these cells confirms that 20 kD hGH is encoded by an alternatively spliced product of the primary hGH-N gene transcript in which the normal exon 3 splice-acceptor site is bypassed for a secondary site 15 codons within exon 3. Although the hGH-V gene is identical to the hGH-N gene for at least 15 nucleotides on either side of the normal and alternative exon 3 AG splice-acceptor sites, hGH-V synthesizes only a 22-kD protein. Reciprocal exchange of exon 3 and its flanking intron sequences between the hGH-N gene and the hGH-V gene, eliminates the synthesis of the 20-kD protein in both resultant chimeric genes. These results directly demonstrate that both the major 22-kD and the minor 20-kD forms of pituitary hGH are encoded by the alternative splicing products of a single hGH-N gene transcript. This alternative splicing is neither species nor tissue-specific and appears to be regulated by at least two separate regions remote from the AG splice-acceptor site.

Multiple response elements in the Sex-lethal early promoter ensure its female-specific expression pattern.

The choice of sexual identity in somatic tissues of the fruit fly Drosophila melanogaster is determined early in embryogenesis by the X-chromosome-to-autosome (X/A) ratio. The system that signals the X/A ratio selects the sexual development pathway by determining the activity state of the binary switch Sex-lethal (Sxl). In 2X/2A animals, the X/A signalling system turns the Sxl gene on, ultimately activating an RNA-splicing autoregulatory feedback loop which serves to maintain the female state during the remainder of development. In 1X/2A animals, this autoregulatory feedback loop is not activated and the male state is subsequently maintained by the default splicing machinery. In the studies reported here, we have examined how the X/A signalling system controls the initial choice of sexual identity through its action on a special early embryonic Sxl promoter, Sxl-Pe. We show that in the early embryo, the activity of Sxl-Pe is controlled in a highly dose-sensitive fashion by the genes on the X chromosome that function as numerator elements and by genes located on the autosomes that function as denominator elements. Functional dissection of Sxl-Pe indicates that activating the promoter in females requires the cumulative action of multiple numerator genes which appear to exert their effects through reiterated cis-acting target sites in the promoter. Conversely, maintaining the promoter silent in males requires the repressive activities of denominator genes, and at least one of the denominator genes also appears to function through target sequences within the promoter.

Interpretation of X chromosome dose at Sex-lethal requires non-E-box sites for the basic helix-loop-helix proteins SISB and daughterless.

For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da) proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding at Sxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter Sxl(Pe). Surprisingly, most SISB-Da sites at Sxl(Pe) differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for Sxl(Pe) activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the Sxl(Pe) switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.

XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer.

The limitations of cancer cell lines have led to the development of direct patient-derived xenograft models. However, the interplay between the implanted human cancer cells and recruited mouse stromal and immune cells alters the tumor microenvironment and limits the value of these models. To overcome these constraints, we have developed a technique to expand human hematopoietic stem and progenitor cells (HSPCs) and use them to reconstitute the radiation-depleted bone marrow of a NOD/SCID/IL2rg(-/-) (NSG) mouse on which a patient's tumor is then transplanted (XactMice). The human HSPCs produce immune cells that home into the tumor and help replicate its natural microenvironment. Despite previous passage on nude mice, the expression of epithelial, stromal and immune genes in XactMice tumors aligns more closely to that of the patient tumor than to those grown in non-humanized mice-an effect partially facilitated by human cytokines expressed by both the HSPC progeny and the tumor cells. The human immune and stromal cells produced in the XactMice can help recapitulate the microenvironment of an implanted xenograft, reverse the initial genetic drift seen after passage on non-humanized mice and provide a more accurate tumor model to guide patient treatment.

Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization.

At an initial step during synaptic vesicle recycling, dynamin and adaptor proteins mediate the endocytosis of synaptic vesicle components from the plasma membrane. StonedA and stonedB, novel synaptic proteins encoded by a single Drosophila gene, have predicted structural similarities to adaptors and other proteins implicated in endocytosis. Here, we test possible roles of the stoned proteins in synaptic vesicle internalization via analyses of third instar larval neuromuscular synapses in two Drosophila stoned (stn) mutants, stn(ts) and stn(8P1). Both mutations reduce presynaptic levels of stonedA and stonedB, although stn(ts) has relatively weak effects. The mutations cause retention of synaptic vesicle proteins on the presynaptic plasma membrane but do not alter the levels or distribution of endocytosis proteins, dynamin, alpha-adaptin, and clathrin. In addition, stn(8P1) mutants exhibit depletion and enlargement of synaptic vesicles. To determine whether these defects arise from altered synaptic vesicle endocytosis or from defects in synaptic vesicle biogenesis, we implemented new methods to assess directly the efficiency of synaptic vesicle recycling and membrane internalization at Drosophila nerve terminals. Behavioral and electrophysiological analyses indicate that stn(ts), an allele with normal evoked release and synaptic vesicle number, enhances defects in synaptic vesicle recycling shown by Drosophila shi(ts) mutants. A dye uptake assay demonstrates that slow synaptic vesicle recycling in stn(ts) is accompanied by a reduced rate of synaptic vesicle internalization after exocytosis. These observations are consistent with a model in which stonedA and stonedB act to facilitate the internalization of synaptic vesicle components from the plasma membrane.

Human progesterone receptor binding to mouse mammary tumor virus deoxyribonucleic acid: dependence on hormone and nonreceptor nuclear factor(s).

Using crude progesterone receptor preparations from T47D human breast cancer cells, we show by immunoprecipitation assay that receptor specifically and with high affinity recognizes the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). The use of crude preparations minimizes alterations of receptors or loss of associated factors that may occur during purification. Specific binding was obtained at 1:1 molar ratios of receptor to DNA, and HRE sequences are recognized with an affinity at least 3 orders of magnitude greater than nonspecific DNA. We have compared the DNA-binding activities of different forms of progesterone receptors. The unliganded 8S cytosol receptor had low but detectable binding activity for MMTV DNA. Addition of hormone to cytosol produced a small but consistent 2.5-fold increase. In vitro methods of transforming cytosol receptors from an 8S to a 4S species failed to increase DNA-binding further. By contrast, 4S receptors bound by R5020 in whole cells and extracted from nuclei by salt, displayed a substantially higher (average, 11-fold) binding activity than an equal number of unliganded cytosol receptors. The dissociation constants for cytosol and nuclear receptor binding to MMTV DNA were similar (approximately 2 x 10(-9) M). Thus, nuclear receptors possess a higher capacity for binding to specific recognition sequences. These results suggest that hormone or a hormone-dependent mechanism increases the intrinsic DNA-binding activity of receptors independent of receptor transformation from 8S to 4S. Further experiments indicate that a nonreceptor activity in nuclear extracts can increase the sequence-specific DNA-binding activity of cytosol receptors. This activity is present in both T47D cells and receptor-negative MDA-231 cells. We conclude that the higher DNA-binding activity of the nuclear receptor-hormone complex is due in part to receptor interaction with other nuclear proteins or factors. Such interactions may function to maintain receptors in a disaggregated active complex or to stabilize their binding to specific DNA sites.

Evidence that heat shock protein-70 associated with progesterone receptors is not involved in receptor-DNA binding.

In the absence of hormone, human progesterone receptors (PR) are recovered in the cytosolic fraction of cell lysates as a multimeric complex containing the steroid-binding polypeptide, heat shock protein-90 (hsp90), and heat shock protein-70 (hsp70). Activated forms of human PR that acquire the ability to bind to DNA are dissociated from hsp90, but retain association with hsp70. The present study has examined whether associated hsp70 has a function in receptor-DNA binding. When activated PR was bound to specific target DNA in a gel shift assay, no hsp70 was detectable in the PR-DNA complex, as evidenced by the failure of several antibodies to hsp70 to affect the mobility or the amount of complexes. To determine whether hsp70 might indirectly influence DNA-binding activity, we have examined the effect of hsp70 dissociation on PR-DNA-binding activity. Dissociation was achieved either by treatment of immunoaffinity-purified immobilized PR complexes with ATP or by the binding of PR complexes to ATP-agarose, followed by elution with high salt. Under both conditions, dissociation from hsp70 neither enhanced nor impaired the ability of PR to bind to specific DNA. These results suggest that hsp70 is not involved in PR binding to DNA, either directly by participating in DNA binding or indirectly by modulating PR-DNA-binding activity. This implies that hsp70 functions at an earlier stage in the receptor activation pathway. Consistent with the known involvement of hsp70 in stabilizing unfolded states of other target proteins, we propose that hsp70 may assist in nuclear transport of PR or in assembly-disassembly of the 8-10S multimeric complex.

Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody.

We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU486. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU486 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody.

Characterization and functional properties of the A and B forms of human progesterone receptors synthesized in a baculovirus system.

Human progesterone receptors (PR) were overexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus system. Recombinant viruses were constructed that produced either full-length A (94K) or B (120K) forms of human PR, and each was expressed as a functional protein. Steroid and DNA binding activities were found to be indistinguishable from that of endogenous human PR in T47D breast cancer cells. Moreover, as analyzed by gel-mobility shift, recombinant PR-A and PR-B each bound to specific progesterone response elements in a strictly hormone-dependent manner. Native receptors expressed in Sf9 cells also exhibited structural properties similar to that of endogenous PR. Cytosolic PR (PR-A or PR-B), prepared in low salt buffer, sedimented on density gradients as an 8S oligomeric complex that was converted largely to 4S by treatment with 0.4 M NaCl. Immune isolation of the 8S cytosol PR complex and analysis of protein composition revealed the presence of two specific copurifying proteins of approximately 90K and 70K. The 90-K component was identified immunologically as heat shock protein 90. The 70-K component was not identified but is likely to be the insect equivalent of heat shock protein 70. Immune isolation of PR from Sf9 cells metabolically labeled with [32Pi], revealed that expressed PR was capable of being phosphorylated in insect cells. Hormone addition to Sf9 cells, however, did not stimulate the same increase in PR phosphorylation or upshift in mobility on sodium dodecyl sulfate gels that occurs with endogenous receptors in T47D cells. Thus some, but not all, phosphorylations occur with human PR expressed in Sf9 cells. These phosphorylation data, together with the fact that expressed PR required hormone for DNA binding, indicate that the hormone-dependent phosphorylation step responsible for PR upshifts on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not required for receptor binding to DNA. The baculovirus expression system, therefore, may prove valuable in dissecting the functional role(s) for both hormone-dependent and hormone-independent PR phosphorylation.

The steroid antagonist RU486 exerts different effects on the glucocorticoid and progesterone receptors.

To determine whether the steroid antagonist RU486 mediates its antiglucocorticoid and antiprogestin activities by the same or different receptor mechanisms, a direct comparison of RU486 interaction with glucocorticoid (GR) and progesterone (PR) receptors was made. The effects of RU486 on transformation of GR and PR 8-10S complexes in the intact cell and in vitro were analyzed by sucrose density gradient centrifugation, and the in vitro stability of receptor-heat shock protein-90 interactions was analyzed by coimmunoprecipitation. Compared to agonist, RU486 binding produced a reduction in the amount of GR converted from 8S to 4S and stabilized the GR-heat shock protein-90 complex. By contrast, PR-RU486 complexes were transformed both in vitro and in the intact cell to the same extent as receptor-agonist complexes. PR-RU486 complexes sedimented at 5-6S, whereas PR-R5020, GR-RU486, and GR-agonist complexes sedimented at 4S. The portion of GR that undergoes nuclear transformation when bound to RU486 was examined for binding to the glucocorticoid-progesterone response element of the mouse mammary tumor virus by an immunoprecipitation assay. The nuclear-transformed GR-RU486 complex bound the glucocorticoid-progesterone response element with the same affinity as the nuclear-transformed GR-triamcinolone acetonide complex. The electrophoretic mobilities of GR-RU486 complexes and GR-agonist complexes were the same, as determined by gel retardation assay. These results suggest that RU486 exerts its antiglucocorticoid activity at two levels of receptor action: prevention of complete GR transformation and alteration of a step subsequent to GR-DNA binding. As an antiprogestin, RU486 action is exerted predominantly at a post-DNA-binding step.

Positive- and negative-acting promoter sequences regulate cell type-specific expression of the rat synapsin I gene.

The phosphoprotein synapsin I is expressed exclusively in neuronal cells. We are interested in elucidating the promoter sequences involved in cell type-specific expression of the synapsin I gene. The PC12 cell line expresses the 3.4 kb and 4.5 kb synapsin I mRNAs and is used to analyze cell type-specific gene expression. A series of deletion fragments of the rat synapsin I gene promoter were fused to the promoterless reporter gene encoding bacterial chloramphenicol acetyltransferase (CAT) for transfection analysis in PC12 cells and in HeLa cells, which do not express the gene. A -349 bp to +110 bp rat synapsin I promoter fragment contains a positive regulator, shown to be 33-times more active in PC12 cells than HeLa cells. Transfection of reporter plasmids containing up to 4.4 kb of rat synapsin I gene promoter sequences exhibit significantly reduced CAT activity in PC12 cells. The reduction in CAT expression was attributed to a negative regulator located between -349 bp and -1341 bp in the rat synapsin I promoter. Our results suggest that both positive and negative-acting sequence elements regulate cell type-specific expression of the rat synapsin I gene.

Mechanisms controlling steroid receptor binding to specific DNA sequences.

Mammalian progesterone receptors activated by hormone binding in nuclei of intact cells exhibit substantially higher binding activity for specific DNA sequences than receptors bound with hormone and activated in cell-free cytosol. Differences in DNA-binding activity occur despite the fact that both activated receptor forms sediment at 4S on sucrose gradients and are apparently dissociated from the heat shock protein 90. This suggests that hormone-induced release of heat shock protein 90 from receptors is necessary, but not sufficient for maximal activation of DNA binding. This report is a review of studies from our laboratories that have examined the role of receptor interaction with other nuclear protein factor(s), and receptor dimerization in solution, as additional regulatory steps involved in the process of receptor activation and binding to specific gene sequences.

Alternative splice site selection in the human growth hormone gene transcript and synthesis of the 20 kDa isoform: role of higher order transcript structure.

Expression of the human growth hormone (hGH) gene in somatotrophs of the anterior pituitary gland results in the synthesis and secretion of a major 22 kDa and a minor 20 kDa GH isohormone. The expression of these two proteins reflects the alternative utilization of a major (B) and a minor (B') splice acceptor site in exon 3 of the hGH-N transcript. By comparing the structure and splicing patterns of the hGH-N gene transcript with that of the structurally related, placentally expressed, hGH-V gene transcript, which uses only the major (B) exon 3 splice acceptor, it has been possible to define the cis-acting elements in exon 3 that are critical for activation of the B' splice acceptor. The present paper demonstrates that, in addition to the importance of sequences in the immediate proximity of the two alternative splice acceptor sites, additional more remote sequences in the transcript also contribute to this alternative splice site selection. The data further suggest that these more distal sequences do not act individually, but interact so that the net level of alternative splicing in exon 3 is dictated by the overall higher order structure of the hGH-N transcript.