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Results for “Genomic characterization”

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PubMed1992

Phenotypic and genetic polymorphisms among human herpesvirus-6 isolates from North American infants.

Fifteen human herpesvirus-6 (HHV-6) isolates from North American infants with primary infection manifest as febrile or roseola (exanthem subitum) like illnesses were characterized phenotypically on the basis of their in vitro growth in continuous T-cell lines and primary human mononuclear cells and by their reactivity with monoclonal antibodies. All isolates replicated efficiently in primary human cord blood mononuclear cells, but five distinct patterns of viral replication in human cells lines were observed. Two of the HHV-6 isolates from infants were found to replicate in HSB-2 cells, a property associated with so-called group A viruses, which had previously been isolated only from adults. These same isolates also reacted with a panel of A-specific monoclonal antibodies. Genomic characterization of viral isolates using well-characterized restriction site polymorphisms indicated that these two isolates contained a mixture of both A- and B-type genomes, in different proportions. These data suggest that not all HHV-6 isolates can be categorized into one of two broad groups and that such segregation of HHV-6 isolates may in fact be misleading.

Cell Line↗

PubMed2026

Genomic and functional characterization of sugar transporters reveals potential roles in sugar accumulation in a modern sugarcane cultivar.

Sugarcane (Saccharum spp.) is a globally important sugar crop whose productivity depends on efficient sugar transport from source to sink organs. However, systematic identification and functional characterization of sugar transporters (STs) in sugarcane cultivars remain limited. Here, we identified 190 non-redundant ST genes in sugarcane cultivar Guitang 42 (GT42) and phylogenetically classified them into nine groups within the Monosaccharide Transporter (MST), Sucrose Transporter (SUT), and Sugars Will Eventually be Exported Transporters (SWEET) families. Comparative evolutionary analysis revealed significant lineage-specific expansions in the PMT, STP subfamilies, and SWEET families compared to diploid and wild relatives, likely driven by polyploidization and intensive selection for sugar yield. Transcriptomic profiling across tissues and internode elongation stages demonstrated marked tissue-specific and developmental expression patterns. Yeast complementation assays confirmed the transport activity of candidate MSTs, SUTs and SWEETs, with confocal microscopy verifying their distinct subcellular localization at the plasma membrane, tonoplast, or endoplasmic reticulum. Furthermore, transient overexpression of several candidate transporters (ScSWEET4-T2, ScSWEET15, and ScTST4-T1) in Nicotiana benthamiana modulated soluble sugar accumulation, and their expression in sugarcane protoplasts activated key sugar-responsive marker genes (ScGPT2 and ScWIP4). Together, our study establishes a systematic genomic framework and identifies candidate functional transporters that govern sugar partitioning and storage, providing valuable genetic targets for molecular breeding and quality enhancement in sugarcane.

Functional characterization↗

PubMed2005

Mitotic polyploidization in trophoblast giant cells of the alpaca.

Genome multiplication is a typical feature of trophoblast giant cell (TGC) development in many species. Elevated nuclear DNA contents can be achieved by modified cell cycles with a complete lack of mitosis (endoreduplication) or with incomplete mitoses. The aim of this study is to characterize genome multiplication in the alpaca TGC. Placental tissues of gestation days 150, 264 and 347 (near term) and term placentae were processed for light microscopy and for transmission electron microscopy. Each TGC showed many nuclear profiles. Observation of serial sections revealed that TGCs are truly multinucleate with several highly lobulated nuclei. Feulgen staining showed that TGC nuclei have a higher DNA content than nuclei of other trophoblast cells. The number of argyrophilic nucleolar organizer regions (AgNORs) in nuclear profiles of TGC was between 15 and 100, while other trophoblast cells showed 1 or 2 AgNORs. Large multipolar mitotic figures with maximal diameters of 80 mum were observed in the alpaca placentas on gestation days 264 and 347. No cytokinesis was seen in TGC. The results show that the mode of genome multiplication in the alpaca TGC is mitotic polyploidization. Subsequent acytokinetic mitoses may lead to an accumulation of chromosomes and centrioles in TGC. With increasing ploidy levels, the shape of these polyploidizing mitoses becomes more irregular. The restitution of nuclei after these complex multipolar mitoses is likely to result in the irregular nuclear shape in TGC.

Animals↗

PubMed1999

[Growing divergence inside primate T cell lymphotropic viruses].

During our studies on the evolution of the PTLVs, we have isolated and genomically characterized two divergent simian T-lymphotropic viruses, not belonging to the previously well-established PTLV lineages. STLV-PH969 has been isolated from an Eritrean sacred baboon (Papio hamadryas), with an HTLV-like indeterminate serological profile. The entire 8916 nt long genomic sequence, was obtained from a cDNA library of PH969 mRNA, in combination with a PCR based strategy. All open reading frames (ORF) common to all HTLV related viruses could be identified without important deletions or insertions. Sequence comparison of the STLV-PH969 genomic sequence with the HTLV-I and -II prototype sequences revealed that this virus is equidistantly related to HTLV-I and -II. A phylogenetic analysis on the envelope and regulatory Tax proteins conclusively proved the ancient separation of HTLV-I, -II and STLV-PH969. Together these data provided enough evidence to justify the classification of this virus as a new type of primate T-lymphotropic virus, designated PTLV-L. We isolated a second highly divergent virus from pygmy chimpanzees (bonobo's, Pan paniscus) housed in the Antwerp Zoo. The animals infected with this virus showed an aberrant HTLV-I like serological profile. The entire 8855 nt long genomic sequence of the STLV-PP1664 provirus was obtained by sequencing of overlapping PCR fragments. On this sequence, all ORFs common to all HTLV related viruses could be identified without any important deletions or insertions. Sequence comparison showed that the STLV-PP1664 proviral sequence was related to HTLV-II and the virus is called STLV-II. However, in all genomic regions, STLV-IIPP1664 was much more divergent from any of the HTLV-II subtypes than these are from each other. In order to further investigate the genomic relationship of STLV-LPH969 and STLV-IIPP1664 with HTLV-I and -II, we also analyzed the genomic organization of the pX region, which differs between HTLV-I and HTLV-II. In the STLV-LPH969 and STLV-IIPP1664 producing cell lines, 2 and 5 viral messengers could be detected respectively, potentially expressing 3 and 5 different accessory proteins respectively. The splicing pattern and the resulting proteins differ from those of HTLV-I and HTLV-II. In the light of the growing interest for xenotransplantation, the prevalence of distinct PTLVs in the wild and their capacity to cross the species barrier deserves further attention.

Animals↗

PubMed2026

Systematic mining and characterization of metal transporter families regulating zinc homeostasis provide insights into metal homeostasis in Camellia sinensis.

BACKGROUND AND AIMS: Zinc is essential for tea plant growth and quality formation, yet its homeostatic mechanisms remain poorly understood. This study identified metal transporter families regulating zinc homeostasis, analyzed their evolution, structure, and expression, and clarified zinc uptake, transport, detoxification networks, and their links to metabolism. METHODS: This study identified zinc homeostasis-related metal transporter families in the tea plant genome, characterized their structural features and expression profiles across tissues and developmental stages through integrative bioinformatics and transcriptomic analyses, and delineated the molecular mechanisms underlying zinc uptake, translocation, and detoxification by systematically integrating published evidence. RESULTS: This study identified 74 metal transporter genes from six families: 13 CsZIPs, 12 CsNRAMPs, 10 CsHMAs, 10 CsYSLs, 14 CsMTPs, and 15 CsCAXs in the 'Shuchazao2' genome, revealing closer affinity to woody species than to Arabidopsis. These proteins exhibit conserved domains, diverse subcellular localizations (cell membrane, vacuole, chloroplast, and Golgi apparatus), and tissue-specific expression with abundant stress/hormone-responsive cis-elements. At the plant-soil interface, tea plants mobilize rhizospheric zinc via proton and organic acid secretion; CsYSLs, CsNRAMPs, and CsZIPs mediate zinc uptake, aided by arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) that expand root absorption zones. Xylem CsHMAs and phloem CsYSLs coordinate root-to-shoot zinc translocation, and vacuolar transporters (CsMTPs, CsCAXs), cell wall immobilization, and antioxidant systems alleviate high-zinc stress injury. CONCLUSIONS: These findings collectively delineate an integrated zinc "acquisition-distribution-buffering" network in tea plants, offering a repertoire of candidate genes with potential utility in zinc biofortification breeding and improving acid soil adaptation. Further experimental validation, including tea transgenesis, zinc-stress qRT-PCR, and heterologous functional complementation, is essential to substantiate their biological roles.

Camellia sinensis↗

PubMed2004

Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome.

We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positivity) by nested-PCR. The eight amplicons obtained by RT-PCR (P1, P3-P9), including one obtained by nested-PCR (P-2) and not obtained by RT-PCR, were sequenced and showed high homology (94.8% to 99.1%) with the N gene of Araraquara hantavirus. Although the serologic method ELISA is the most appropriate test for HCPS diagnosis, the use of nested RT-PCR for hantavirus in Brazil would contribute to the diagnosis of acute hantavirus disease detecting viral genomes in patient specimens as well as initial genomic characterization of circulating hantaviruses.

Adolescent↗

PubMed2006

Genomic organization of a diverse ACC synthase gene family in banana and expression characteristics of the gene member involved in ripening of banana fruits.

The banana is one of the typical climacteric fruits with great economic importance in agriculture. To understand the basic mechanism underlying banana ripening, gene clones for banana ACC synthase (EC 4.4.1.14), a key regulatory enzyme in the ethylene biosynthetic pathway, were characterized. Genomic clones were analyzed by restriction mapping, and the data in conjunction with sequence comparisons with the previously isolated PCR fragments indicated that at least nine ACC synthase genes (MACS1-9) exist in the banana genome. Southern blot analysis showed they are located in different regions of the banana genome. Three lambda genomic clones (GMACS-1, -9, and -12) were completely sequenced, and gene structures of MACS1 (corresponding to alleles of GMACS-9 and -12) and MACS2 (corresponding to GMACS-1) were elucidated. The coding regions of these three genes were all interrupted by three introns. The size and location of introns are similar to the ACC synthase genes from tomato and Arabidopsis. Northern analysis showed that only MACS1 expressed during fruit ripening and was inducible by exogenous ethylene treatment, which indicates MACS1 is a significant member of the ACC synthase gene family related to ripening in banana fruit. The transcription initiation site of GMACS-12 containing MACS1 was defined. There is a TATTAAT sequence located at position -31 to -25 that qualifies as a TATA box. The delineation of transcription unit in MACS1 will facilitate the promoter studies for this gene and allow its specific functions involved in fruit ripening to be determined.

Amino Acid Sequence↗

PubMed1996

Lymphomatoid papulosis and cutaneous CD30+ lymphoma.

Lymphomatoid papulosis and cutaneous CD30+ lymphoma are closely related conditions in which large atypical lymphocytes that have similar immunophenotypic features occur. In lymphomatoid papulosis, the lesions are papules and nodules that spontaneously involute. There are two polar histologic patterns, type A and B, in which the large atypical cells resemble those of Hodgkin's disease and mycosis fungoides, respectively, but in many cases, features of both types are present, either separately or in the same lesions. Variants of lymphomatoid papulosis include cases with a perifollicular distribution and those with lymphocytic vasculitis or dermal mucin deposits. Clinical lesions that tend to be stable, a monomorphous cellular composition, and in the case of immunocompromised patients, the presence of Epstein-Barr viral genome characterize cutaneous CD30+ lymphoma. A loss of response to transforming growth factor-beta, which normally dampens cellular proliferation, may differentiate CD30+ lymphoma from lymphomatoid papulosis.

Cell Division↗

PubMed2004

Genomic patterns of allelic imbalance in disease free tissue adjacent to primary breast carcinomas.

Mammary stroma plays an important role in facilitating the neoplastic transformation of epithelial cells, modulating integrity of the extracellular matrix, and maintaining genomic stability, but molecular mechanisms by which stroma affects epithelial structure and function are not well-defined. We used laser-assisted microdissection of paraffin-embedded breast tissues from 30 patients with breast disease and a panel of 52 microsatellite markers defining 26 chromosomal regions to characterize genomic patterns of allelic imbalance (AI) in disease-free tissue adjacent to sites of breast disease and to define genomic regions that may contain genes associated with early carcinogenic processes. The mean frequency of AI in histologically normal tissue adjacent to the primary carcinomas (15.4%) was significantly higher than that in distant tissue from the same breast (3.7%). The pattern of AI across all chromosomal regions differed between the adjacent tissue and primary tumor in every case. Unique AI events, observed only in tumor (15% of informative markers) or only in adjacent cells (10% of informative markers), were far more common than AI events shared between tumor and adjacent cells (approximately 4%). Levels of AI characteristic of advanced invasive carcinomas were already present in non-invasive ductal carcinomas in situ, and appreciable levels of AI were observed in adjacent non-neoplastic tissue at all pathological stages. Chromosome 11p15.1 showed significantly higher levels of AI in adjacent cells (p < 0.01), suggesting that this region may harbor genes involved in breast cancer development and progression. Our data indicate that genomic instability may be inherently greater in disease-free tissue close to developing tumors, which may have important implications for defining surgical margins and predicting recurrence.

Adult↗

PubMed2000

Preliminary profile of the Cryptosporidium parvum genome: an expressed sequence tag and genome survey sequence analysis.

Cryptosporidium parvum is a protozoan enteropathogen that infects humans and animals and causes a pronounced diarrheal disease that can be life-threatening in immunocompromised hosts. No specific chemo- or immunotherapies exist to treat cryptosporidiosis and little molecular information is available to guide development of such therapies. To accelerate gene discovery and identify genes encoding potential drug and vaccine targets we constructed sporozoite cDNA and genomic DNA sequencing libraries from the Iowa isolate of C. parvum and determined approximately 2000 sequence tags by single-pass sequencing of random clones. Together, the 567 expressed sequence tags (ESTs) and 1507 genome survey sequences (GSSs) totaled one megabase (1 mb) of unique genomic sequence indicating that approximately 10% of the 10.4 mb C. parvum genome has been sequence tagged in this gene discovery expedition. The tags were used to search the public nucleic acid and protein databases via BLAST analyses, and 180 ESTs (32%) and 277 GSSs (18%) exhibited similarity with database sequences at smallest sum probabilities P(N)< or =10(-8). Some tags encoded proteins with clear therapeutic potential including S-adenosylhomocysteine hydrolase, histone deacetylase, polyketide/fatty-acid synthases, various cyclophilins, thrombospondin-related cysteine-rich protein and ATP-binding-cassette transporters. Several anonymous ESTs encoded proteins predicted to contain signal peptides or multiple transmembrane spanning segments suggesting they were destined for membrane-bound compartments, the cell surface or extracellular secretion. One-hundred four simple sequence repeats were identified within the nonredundant sequence tag collection with (TAA)(> or =6)/(TTA)(> or =6) and (TA)(> or = 10)/(AT)(> or =10 ) being the most prevalent, occurring 40 and 15 times, respectively. Various cellular RNAs and their genes were also identified including the small and large ribosomal RNAs, five tRNAs, the U2 small nuclear RNA, and the small and large virus-like, double-stranded RNAs. This investigation has demonstrated that survey sequencing is an efficient procedure for gene discovery and genome characterization and has identified and sequence tagged many C. parvum genes encoding potential therapeutic targets.

Amino Acid Sequence↗

PubMed2005

Shotgun haplotyping: a novel method for surveying allelic sequence variation.

Haplotypic sequences contain significantly more information than genotypes of genetic markers and are critical for studying disease association and genome evolution. Current methods for obtaining haplotypic sequences require the physical separation of alleles before sequencing, are time consuming and are not scaleable for large surveys of genetic variation. We have developed a novel method for acquiring haplotypic sequences from long PCR products using simple, high-throughput techniques. This method applies modified shotgun sequencing protocols to sequence both alleles concurrently, with read-pair information allowing the two alleles to be separated during sequence assembly. Although the haplotypic sequences can be assembled manually from the resultant data using pre-existing sequence assembly software, we have devised a novel heuristic algorithm to automate assembly and remove human error. We validated the approach on two long PCR products amplified from the human genome and confirmed the accuracy of our sequences against full-length clones of the same alleles. This method presents a simple high-throughput means to obtain full haplotypic sequences potentially up to 20 kb in length and is suitable for surveying genetic variation even in poorly-characterized genomes as it requires no prior information on sequence variation.

Algorithms↗

PubMed2004

One hundred fifty-four genetic markers for the turkey (Meleagris gallopavo).

Identifying and selectively breeding for improved traits is one of the ultimate goals of genetic research in agriculturally important species. Genome characterization and analysis are important first steps in this process. Genetic linkage maps based on the linear order of polymorphic DNA markers are typically developed through statistical analysis of inheritance patterns in pedigreed families. To develop microsatellite markers for further improvement of the turkey genetic linkage map, small-insert genomic libraries were screened for tandem repeats. Oligonuclotide primers were designed to amplify 164 microsatellite-containing fragments from genomic DNA. Genetic polymorphisms at 154 markers were determined by genotyping the F(1) individuals of two resource populations. Markers determined as segregating in the University of Minnesota/Nicholas Turkey Breeding Farms (UMN/NTBF) reference population were used to genotype F(2) individuals and a two-point linkage analysis was performed.

Alleles↗

PubMed2026

A Clinically Integrated Pediatric Patient-Derived Xenograft Program Enables Evaluation of Cohort and Patient-Specific Biology and Therapeutic Strategies.

UNLABELLED: Preclinical translational research has increasingly utilized patient-derived xenograft (PDX) models for mechanistic and experimental therapeutic studies, yet most existing models have been developed from adult cancer types. We describe the establishment of a PDX program to expand the availability of pediatric-specific PDXs for preclinical research and enable studies of pediatric cancer histologies, including ultrarare diseases. Processes for PDX generation were integrated into established clinical workflows to facilitate universal model generation. Methodologies for tissue procurement, processing, and cryopreservation were optimized to enable intra- and interinstitutional PDX model generation. Over a 6-year span, 388 PDX tumor models representing more than 40 diagnoses were generated, including ultrarare tumors and longitudinal models established from pretherapy, posttherapy, and relapse tumors from the same patient. Genomic characterization of these PDXs demonstrates excellent concordance and recapitulation of molecular alterations of the source tumor. Successful PDX generation was enhanced from relapsed samples, was higher in sarcomas compared with other solid tumor types, and was a negative prognosticator for clinical outcome. With a broad portfolio of molecularly annotated models, we demonstrate utility for validating cross-histology biomarker-driven therapeutic strategies by demonstrating antitumor activity of an MAT2A inhibitor in MTAP-deficient PDXs. Universal model creation also allows for experimental validation of therapeutic hypotheses on a patient-specific basis, as we describe the characterization of a novel RAF1 fusion (EPB41L2::RAF1) in an osteosarcoma PDX. Development of a diverse collection of pediatric PDX models enables hypothesis-driven and cross-histology studies that expand our understanding of cancer biology and aid ongoing drug prioritization efforts in rare tumors. SIGNIFICANCE: A clinically integrated, genomically annotated pediatric PDX portfolio supported by systematic benchmarking of model generation facilitates exploratory biomarker-driven and patient-specific translational studies.

Humans↗

PubMed1994

Subcellular distribution and characterization of glucosephosphate isomerase in Leishmania mexicana mexicana.

The glycolytic enzyme glucosephosphate isomerase (PGI) is present in two different cell compartments of Leishmania mexicana promastigotes; more than 90% of the activity was detected in the cytosol, the remainder in glycosomes. This subcellular distribution contrasts with that in Trypanosoma brucei, in which the enzyme activity has been mainly located in the glycosomes. PGI was partially purified from L. mexicana cell extracts. Throughout the purification procedure only one single PGI activity could be detected. The partially purified protein had the same subunit molecular mass (65 kDa) as the previously characterized glycosomal protein of T. brucei. Both proteins were also very similar with respect to their kinetic and antigenic properties. Using the T. brucei glycosomal PGI gene as a hybridization probe, we cloned the corresponding gene of L. mexicana. Only a single PGI locus could be detected in the L. mexicana genome. Characterization of the cloned gene showed that it codes for a polypeptide of 604 amino acids, with a molecular mass of 67,113. The sequences of the Leishmania and Trypanosoma polypeptides are 69% identical. They differ in calculated net charge (-8 versus -2, respectively) and isoelectric point (6.65 versus 7.35). Our data strongly suggest that the PGI activity in the two cell compartments of L. mexicana and T. brucei is not attributable to different isoenzymes. We discuss the possible metabolic function of the highly different enzyme distribution in the two organisms, and the molecular mechanism that could be responsible for it.

Amino Acid Sequence↗

PubMed1996

The human interleukin-11 receptor alpha gene (IL11RA): genomic organization and chromosome mapping.

The high-affinity receptor for interleukin-11 (IL-11) is composed of two subunits, IL-11 receptor alpha chain (IL-11R alpha) and gp130, the common subunit of the interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, and oncostatin M receptors. The IL-11 receptor-specific alpha chain shares homologies with the alpha chain of the CNTF and IL-6 receptors. We isolated and characterized genomic DNA clones encompassing the entire coding sequence of the IL-11R alpha cDNA. The exon-intron organization of the IL-11R gene (HGMW-approved symbol IL11RA) is consistent with the predicted structure of the different domains of the IL-11R alpha protein, confirming evolutionary conservation at the level of gene organization among the hematopoietic cytokine receptor family. The IL-11R gene has been assigned to chromosome 9 band p13 by in situ hybridization using human IL-11R alpha cDNA as a probe. The fact that the ciliary neurotrophic factor (CNTFR) gene has recently been localized on this same band and the conserved genomic structure between IL-11R and CNTFR suggest that they may have evolved from a common ancestor.

Base Sequence↗

PubMed1997

Adenovirus-mediated gene transfer in neurons: construction and characterization of a vector for heterologous expression of the axonal cell adhesion molecule axonin-1.

By homologous recombination, a first-generation adenovirus-based gene transfer vector, AdCMVax-1, was constructed as a means of manipulating the expression level of the axonal cell adhesion molecule axonin-1 in neurons and glial cells. AdCMVax-1 harbours the entire coding region of the chicken axonin-1 cDNA under the transcriptional control of the Cytomegalovirus enhancer/promoter in the early-region 1 of the viral genome. Characterization of AdCMVax-1 in vitro revealed highly efficient gene transfer and expression of recombinant axonin-1 in neurons and glial cells of dissociated rat dorsal root ganglia. Similar to its native counterpart, virus-derived axonin-1 was detected on the cell body, neurites, and growth cones of transduced neurons, occurred in a secreted and membrane-associated form, and could be cleaved from the membrane with phosphatidylinositol-specific phospholipase C. Functional characterization of recombinant axonin-1 revealed the same binding properties as previously reported for native axonin-1 isolated from the vitreous fluid of chicken embryos. In vivo gene transfer was studied by stereotactic injection of AdCMVax-1 in the dentate gyrus of the hippocampus and the facial nucleus in the brainstem of adult Wistar rats and revealed high level expression of recombinant axonin-1 in a subset of hippocampal neurons and motor neurons in the facial nucleus.

Adenoviruses, Human↗

PubMed2026

Draft genome sequence of Enterococcus casseliflavus strain MBBL_MP4 isolated from healthy bovine milk.

We report the draft genome sequence of Enterococcus casseliflavus MBBL_MP4, recovered from healthy bovine milk. The 3.45-Mbp genome assembly comprises 27 contigs and indicates low pathogenic potential, with no acquired antimicrobial resistance or known virulence genes. This genome provides a valuable resource for the genomic characterization of bovine-associated E. casseliflavus.

Enterococcus casseliflavus↗

PubMed1993

Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes.

PCR of cDNA produced from patient fibroblasts allowed us to determine the paternal mutation in the first patient reported with beta-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G-->T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reduced stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, we found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed us to amplify and characterize genomic sequences for the true beta-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C-->T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression, suggesting that high concentrations of mutant monomers can drive the folding and tetramerization of mutant enzyme to produce an active and stable enzyme.

Animals↗