Variable loss of functional activities of androgen receptor mutants in patients with androgen insensitivity syndrome.

Androgen receptor (AR) mutations in androgen insensitivity syndrome (AIS) are associated with a variety of clinical phenotypes. The aim of the present study was to compare the molecular properties and potential pathogenic nature of 8 novel and 3 recurrent AR variants with a broad variety of functional assays. Eleven AR variants (p.Cys177Gly, p.Arg609Met, p.Asp691del, p.Leu701Phe, p.Leu723Phe, p.Ser741Tyr, p.Ala766Ser, p.Arg775Leu, p.Phe814Cys, p.Lys913X, p.Ile915Thr) were analyzed for hormone binding, transcriptional activation, cofactor binding, translocation to the nucleus, nuclear dynamics, and structural conformation. Ligand-binding domain variants with low to intermediate transcriptional activation displayed aberrant Kd values for hormone binding and decreased nuclear translocation. Transcriptional activation data, FxxFF-like peptide binding and DNA binding correlated well for all variants, except for p.Arg609Met, p.Leu723Phe and p.Arg775Leu, which displayed a relatively higher peptide binding activity. Variants p.Cys177Gly, p.Asp691del, p.Ala766Ser, p.Phe814Cys, and p.Ile915Thr had intermediate or wild type values in all assays and showed a predominantly nuclear localization in living cells. All transcriptionally inactive variants (p.Arg609Met, p.Leu701Phe, p.Ser741Tyr, p.Arg775Leu, p.Lys913X) were unable to bind to DNA and were associated with complete AIS. Three variants (p.Asp691del, p.Arg775Leu, p.Ile915Thr) still displayed significant functional activities in in vitro assays, although the clinical phenotype was associated with complete AIS. The data show that molecular phenotyping based on 5 different functional assays matched in most (70%) but not all cases.

Functional analysis of novel androgen receptor mutations in a unique cohort of Indonesian patients with a disorder of sex development.

Mutations in the androgen receptor (AR) gene, rendering the AR protein partially or completely inactive, cause androgen insensitivity syndrome, which is a form of a 46,XY disorder of sex development (DSD). We present 3 novel AR variants found in a cohort of Indonesian DSD patients: p.I603N, p.P671S, and p.Q738R. The aim of this study was to determine the possible pathogenic nature of these newly found unclassified variants. To investigate the effect of these variants on AR function, we studied their impact on transcription activation, AR ligand-binding domain interaction with an FxxLF motif containing peptide, AR subcellular localization, and AR nuclear dynamics and DNA-binding. AR-I603N had completely lost its transcriptional activity due to disturbed DNA-binding capacity and did not show the 114-kDa hyperphosphorylated AR protein band normally detectable after hormone binding. The patient with AR-I603N displays a partial androgen insensitivity syndrome phenotype, which is explained by somatic mosaicism. A strongly reduced transcriptional activity was observed for AR-Q738R, together with diminished interaction with an FxxLF motif containing peptide. AR-P671S also showed reduced transactivation ability, but no change in DNA- or FxxLF-binding capacity and interferes with transcriptional activity for as yet unclear reasons.

RET and GDNF gene scanning in Hirschsprung patients using two dual denaturing gel systems.

Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal obstruction due to an absence of intramural ganglia along variable lengths of the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF gene, and encoding one of the RET ligands, either alone or in combination with RET mutations, can also cause HSCR, as can mutations in four other genes (EDN3, EDNRB, ECE1, and SOX10). The rare mutations in the latter four genes, however, are more or less restricted to HSCR associated with specific phenotypes. We have developed a novel comprehensive mutation detection system to analyse all but three amplicons of the RET and GDNF genes, based on denaturing gradient gel electrophoresis. We make use of two urea-formamide gradients on top of each other, allowing mutation detection over a broad range of melting temperatures. For the three remaining (GC-rich) PCR fragments we use a combination of DGGE and constant denaturing gel electrophoresis (CDGE). These two dual gel systems substantially facilitate mutation scanning of RET and GDNF, and may also serve as a model to develop mutation detection systems for other disease genes. In a screening of 95 HSCR patients, RET mutations were found in nine out of 17 familial cases (53%), all containing long segment HSCR. In 11 of 78 sporadic cases (14%), none had long segment HSCR. Only one GDNF mutation was found, in a sporadic case.

Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria.

Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared from fecal samples of three healthy volunteers. To introduce variation between the samples, they were incubated for 48 h in batch culture (anaerobic) fermenters at 37 degrees C, and pure cultures of Bifidobacterium infantis, Clostridium perfringens, or Lactobacillus acidophilus were added. Samples were taken from the fermenters at different times. Total anaerobes, bifidobacteria, bacteroides, clostridia, and lactobacilli were enumerated by both plating and FISH. The results showed that plate counts of total anaerobes, bifidobacteria, lactobacilli and bacteroides were approximately ten-fold lower than the corresponding FISH counts. Numbers of clostridia were higher using the plating method, probably because the clostridia probe used in FISH analyses was designed to only detect part of the genus Clostridium. The introduced variation in the methods could be detected by both methods and was comparable.

Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is believed to be the most powerful pre-screening method for mutation detection currently available, being used mostly on an exon-by-exon basis. Broad-range DGGE for the analysis of multiple fragments or an entire gene is rarely applied. We and others have already shown that one or two DGGE conditions are usually sufficient to analyse an entire gene. Conditions, however, have never been profoundly tested and compared with alternative methods suggested in the literature. Trying to do so in this study, we found significant differences between the various gel systems. The optimal conditions we found for broad-range DGGE include 9% polyacrylamide for the gel, a denaturing gradient with a difference of 30-50% between the lowest and the highest concentration of denaturant, and electrophoresis in 0.5x TAE buffer at a voltage >100 V and <200 V.

Improved mutation detection in GC-rich DNA fragments by combined DGGE and CDGE.

Denaturing gradient gel electrophoresis (DGGE) has proven to be a powerful pre-screening method for the detection of DNA variants. If such variants occur, however, in DNA fragments that are very rich in G and C, they may escape detection. To overcome this limitation, we tested a novel gel system which combines DGGE and constant denaturant gel electrophoresis (CDGE), as it might have the advantages of both methods. Indeed, this combination had the advantages of both methods, good separation of hetero-duplex molecules and prevention of total strand dissociation, and it proved successful in the detection of DNA variants in several GC-rich fragments.

16S ribosomal RNA-targeted oligonucleotide probes for monitoring of intestinal tract bacteria.

BACKGROUND: The composition of a sample of faecal bacteria can be determined by culturing different dilutions on specific media. However, not all bacteria can be cultured and media are not always specific. With a culture-independent approach a more accurate picture of the composition of the intestinal flora may be obtained. METHODS: Fluorescently labelled oligonucleotide probes targeted at 16S ribosomal RNA sequences specific for a bacterial genus were designed and applied for fluorescence in situ hybridization (FISH) of bacteria in human faecal samples. RESULTS: The mean number of Bifidobacterium spp. and the total number of anaerobic bacteria per gram of faeces were determined by culturing and with the probe technique. Although in both cases the number of Bifidobacterium spp. was about the same, 2.38 x 10(9) and 2.45 x 10(9), it was found that the contribution of Bifidobacterium spp. to the total composition is overestimated due to the lower number of total anaerobic bacteria estimated by culturing. CONCLUSION: Genus-specific or group-specific fluorescent 16S rRNA probes may become an invaluable tool in gut ecology studies.

Altered AP-1/ATF complexes in adenovirus-E1-transformed cells due to EIA-dependent induction of ATF3.

The adenovirus (Ad) E1A proteins alter the expression level and activity of AP-1/ATF transcription factors. Previously we have shown that in AdE1-transformed cells cJun is hyperphosphorylated in its N-terminal transactivation domain, which parallels enhanced transactivation function. To find out whether the interaction between cJun and other cellular proteins is altered, we have searched for proteins which would physically associate with cJun. In this report we show that in AdE1-transformed cells cJun specifically associates with two proteins of 21 and 23 kD. These proteins are not expressed at detectable levels in the parental cells or in cells transformed by oncogenes other than AdE1. The cJun-associated proteins represent different forms of the bZIP transcription factor ATF3, the human homolog of rat LRF1. The expression of ATF3 is induced in AdE1-transformed cells and is a direct effect of the expression of E1A. Through induction of ATF3 expression and the subsequent formation of cJun/ATF3 heterodimers, E1A alters the repertoire of AP-1/ATF factors and may thereby redirect the corresponding gene-expression program. Since the induction of ATF3 is a function of sequences within the transforming 12S-ElA protein, cJun/ATF3 complexes might be involved in establishing cellular transformation by AdE1A.

The N-terminal region of the adenovirus type 5 E1A proteins can repress expression of cellular genes via two distinct but overlapping domains.

The transforming E1A 12S and E1A 13S proteins of human adenovirus type 5 (Ad5) contain two and three conserved regions, respectively. In the present study, the contribution of sequences in the nonconserved N-terminal region of the E1A proteins to morphological transformation and to down-regulation of a number of mitogen-inducible genes was investigated. As described previously, transformation of NRK cells (an established normal rat kidney cell line) results in denser cell growth and a cuboidal cellular morphology. None of the cells expressing N-terminally mutated E1A proteins, however, show these transformed properties, which suggests an important role for sequences in that domain. The decrease in cyclin D1 levels requires exactly the same sequences. The ability to transform NRK cells and to reduce cyclin D1 levels does not correlate with the presence in the E1A proteins of binding domains for p300, CBP, p107, pRb, cyclin A, or cdk2. In contrast, down-regulation of expression of the JE gene in NRK cells and repression of transcription of the collagenase gene in human HeLa cells does correlate with the presence in the E1A proteins of an intact binding domain for p300 and CBP. The results suggest that the N-terminal domain of the E1A proteins can repress expression of cellular genes by at least two different mechanisms.

Rapid c-myc mRNA degradation does not require (A + U)-rich sequences or complete translation of the mRNA.

The c-myc proto-oncogene encodes a highly unstable mRNA. Stabilized, truncated myc transcripts have been found in several human and murine tumors of hematopoietic origin. Recently, two tumors expressing 3' truncated c-myc mRNAs that were five times more stable than normal myc transcripts, were described. We have tried to determine the cause of the increased stability of the 3' truncated myc transcripts by studying the half-life of mutated c-myc mRNAs. The c-myc 3' untranslated region has been shown to contain sequences that confer mRNA instability. Possible candidates for such sequences are two (A + U)-rich regions in the 3' end of the c-myc RNA that resemble RNA destabilizing elements present in the c-fos and GMCSF mRNAs. We show that deletions in the (A + U)-rich regions do not stabilize c-myc messengers, and that hybrid mRNAs containing SV40 sequences at their 3' ends and terminating at an SV40 polyadenylation signal decay as quickly as normal c-myc transcripts. Our results indicate that neither the loss of (A + U)-rich sequences nor the mere addition of non-myc sequences to the 3' end of the mRNA lead to stabilization. We also show that rapid degradation of c-myc mRNA does not require complete translation of the coding sequences.

Poly(A) tail shortening is the translation-dependent step in c-myc mRNA degradation.

The highly unstable c-myc mRNA has been shown to be stabilized in cells treated with protein synthesis inhibitors. We have studied this phenomenon in an effort to gain more insight into the degradation pathway of this mRNA. Our results indicate that the stabilization of c-myc mRNA in the absence of translation can be fully explained by the inhibition of translation-dependent poly(A) tail shortening. This view is based on the following observations. First, the normally rapid shortening of the c-myc poly(A) tail was slowed down by a translation block. Second, c-myc messengers which carry a short poly(A) tail, as a result of prolonged actinomycin D or 3'-deoxyadenosine treatment, were not stabilized by the inhibition of translation. We propose that c-myc mRNA degradation proceeds in at least two steps. The first step is the shortening of long poly(A) tails. This step requires ongoing translation and thus is responsible for the delay in mRNA degradation observed in the presence of protein synthesis inhibitors. The second step involves rapid degradation of the body of the mRNA, possibly preceded by the removal of the short remainder of the poly(A) tail. This last step is independent of translation.

RAS gene mutations in childhood acute myeloid leukemia: a Pediatric Oncology Group study.

Mutations at codon 12, 13, and 61 of the HRAS, KRAS, and NRAS genes were evaluated in 99 cases of pediatric acute myeloid leukemia (AML) using oligonucleotide hybridization to polymerase chain reacted derived products. Twenty-four mutations were identified in the NRAS gene, 13 in the KRAS gene, and none in the HRAS gene. The mutations occurred in a broad spectrum of cases, and there was no specific association of RAS gene mutations with patient subsets defined on the basis of clinical or hematologic features. These data demonstrate that RAS gene mutations are at least as common in childhood AML as in adult AML and suggest that RAS gene mutations play a role in myeloid neoplasia in both age groups.

Analysis of polyadenylation site usage of the c-myc oncogene.

The c-myc gene contains 2 well conserved polyadenylation (pA) sites. In all human and rat cell lines from various differentiation stages and tissue types the amount of mRNA terminating at the second pA site is 6-fold higher than the amount ending at the upstream site. This is not due to a difference in stability of the two mRNA types and therefore must be due to preferential usage of the downstream site. The usage of the pA sites is not altered during growth factor induction of quiescent cells. We have not been able to detect differences in behavior between mRNAs ending at either pA site. Both types of mRNA are induced upon treatment of cells with cycloheximide. Furthermore, we have shown that the poly(A) tail of c-myc mRNA is lost during degradation of the messenger, as was described previously for c-myc mRNA in an in vitro system. The time required for the loss of the poly(A) tail is similar to the half-life of c-myc mRNA.