Biomedical subjects

M Tudor

Publications and source records attributed to M Tudor.

At least 19 recordsLinked to original sources

PubMed2002

A 5.9-kb tandem repeat at the euchromatin-heterochromatin boundary of the X chromosome of Drosophila melanogaster.

We present an analysis of a chromosomal walk in the region of the euchromatin-heterochromatin transition at the base of the X chromosome of Drosophila melanogaster. This region is difficult to analyse because of the presence of repeated sequences, and we have used cosmids to walk from the last euchromatic gene, suppressor of forked, towards the pericentric heterochromatin. The proximal 30-kb sequence we have isolated consists of repetitive DNA, including four tandem copies of a 5.9-kb sequence. This tandem repeat is itself a mosaic of other, mostly repeated, sequences, including part of a retrotransposon without long terminal repeats, a simple-sequence region of TAA repeats and part of a retrotransposon with long terminal repeats that has not been previously described. Although sequences homologous to these components are found elsewhere in the genome, this arrangement of repeated sequences is only found at the base of the X chromosome. It is conserved in D. melanogaster strains of different geographic origin, but is not conserved in even closely related species.

Animals↗

PubMed2001

Yeast artificial chromosome targeting technology: an approach for the deletion of genes in the C57BL/6 mouse.

An approach is described to modify yeast artificial chromosomes (YACs) with cassettes that can be easily excised for embryonic stem (ES) cell gene targeting experiments. YAC targeting technology (YTT) uses the WIBR/MIT-820 C57BL/6-mapped YAC library derived from the C57BL/6 mouse as the starting point for Internet- or PCR-based clone isolation, although in principle any YAC system can be used. Homologous recombination is initially performed in yeast using cassettes that function in Saccharomyces cerevisiae, Escherichia coli, and ES cells, followed by cloning or conversion of the targeted locus into a plasmid. The completed targeting vector can be transfected into C57BL/6 ES cells and clones selected with G418 followed by injection into Balb/c blastocysts. YTT increases the speed of targeting vector construction and obviates the need for extensive backcrossing to the C57BL/6 background.

Animals↗

PubMed2001

[Penetrating combat injuries of the colorectal region].

INTRODUCTION: Colorectal war injuries can be treated with primary repair or by colostomy. We report our experience with both treatments. METHODS: During the Croatian war from July 1991 to March 1994, 155 patients with colorectal injuries were treated at the Surgical Clinic, University Hospital Split. This group represents 7% of all patients (n = 2220) with gunshot and shrapnel wounds treated in this period at our clinic. The median patient age was 24.7 years (range 14-70 years). The majority of the patients (96.7%) were male. Concomitant injuries of the colon (83.7%) and rectum (69%) were found more frequently than isolated ones. 50 patients were operated on as emergencies in our clinic, while 105 were operated on in field hospitals. In 28 patients primary repair of the colorectal injury was performed (without derivation), whereas 127 patients were treated by colostomy. In those patients 106 wounds were closed electively during a second operation. The average in hospital stay was 32.3 days (range 10-65 days). RESULTS: Using PATI and FCIS scores for colorectal injuries, 80% of our patients had life-threatening injuries. In the cases with primary repair the percentage of complications was high (92%). In the cases with the diverting colostomy it was only 34%. The high complication rate in the cases with primary repair was directly related to the presence of the anastomotic leaks and subsequent peritonitis. Explorative laparotomy was an effective diagnostic tool especially in the field hospitals. The overall mortality rate was 3.2%. CONCLUSION: In our operative strategy we preferred derivation operations in order to decrease major complications due to anastomotic leakage or peritonitis.

Adolescent↗

PubMed2001

The X element, a novel LINE transposable element from Drosophila melanogaster.

Whilst analysing the nature of repeated DNA sequences in the transition zone between euchromatin and heterochromatin at the base of the X chromosome of Drosophila melanogaster, we discovered a novel transposable element of the LINE class that we have named the X element. Several apparently complete elements have been cloned and analysed, and one has been sequenced. It is 4740 bp long, with a polyadenylation sequence and a run of A residues at one end. It contains two ORFs: the 5' ORF is related to the retroviral gag gene and encodes a protein with cysteine-rich motifs that are thought to form a "zinc-knuckle" in a nucleic-acid binding protein; the 3' ORF encodes a putative reverse transcriptase that includes the conserved domains found in reverse transcriptases from other LINEs and retroviruses. The DNA sequence and the sequences of the predicted gene products are most similar to other LINEs from D. melanogaster, such as the F, jockey, Doc and BS elements. Southern analysis suggests that there are at least 30 copies in the genome and that some elements are polymorphic between different strains. Analysis of the DNA sequence of the euchromatic arms of the Drosophila genome identified five full-length elements and a similar number of elements that were intact at the 3' end but had variable 5' truncations. Sequences flanking two different insertion sites were used to design PCR primers to assess the occupancy of sites in wild-type flies of different geographical origins. Flies that lacked each of the insertions were found, suggesting that the element is an active transposon.

Animals↗

PubMed2001

Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation.

Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.

Alleles↗

PubMed2001

Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice.

Mecp2 is an X-linked gene encoding a nuclear protein that binds specifically to methylated DNA (ref. 1) and functions as a general transcriptional repressor by associating with chromatin-remodeling complexes. Mecp2 is expressed at high levels in the postnatal brain, indicating that methylation-dependent regulation of gene expression may have a crucial role in the mammalian central nervous system. Consistent with this notion is the recent demonstration that MECP2 mutations cause Rett syndrome (RTT, MIM 312750), a childhood neurological disorder that represents one of the most common causes of mental retardation in females. Here we show that Mecp2-deficient mice exhibit phenotypes that resemble some of the symptoms of RTT patients. Mecp2-null mice were normal until 5 weeks of age, when they began to develop disease, leading to death between 6 and 12 weeks. Mutant brains showed substantial reduction in both weight and neuronal cell size, but no obvious structural defects or signs of neurodegeneration. Brain-specific deletion of Mecp2 at embryonic day (E) 12 resulted in a phenotype identical to that of the null mutation, indicating that the phenotype is caused by Mecp2 deficiency in the CNS rather than in peripheral tissues. Deletion of Mecp2 in postnatal CNS neurons led to a similar neuronal phenotype, although at a later age. Our results indicate that the role of Mecp2 is not restricted to the immature brain, but becomes critical in mature neurons. Mecp2 deficiency in these neurons is sufficient to cause neuronal dysfunction with symptomatic manifestation similar to Rett syndrome.

Animals↗

PubMed2001

Unusual stab wound of the temporal region.

We report the case of an unusual penetrating injury of the temporal region of the head caused by knife. A long kitchen knife was protruding from the upper auricular area of the left temporal region of victims head. It cut through the posterosuperior part of the left auricula and remained fixed to the postauricular region. Brain computorized tomography (CT) scan revealed that the knife had reached deep into the petrous part of the temporal bone, and was directed toward the sulcus of the sigmoid sinus. There were no signs of intracranial bleeding. An otorhinolaryngologist and a neurosurgeon removed the knife in operating room, with the patient in general anesthesia. The audiogram obtained after 7 days of hospitalization showed left conductive hearing loss of 40 dB at frequencies up to 2 kHz, and of 90 dB above 2 kHz, probably due to hemotympanum caused by the operation. Control examinations performed 14 days and one month after discharge confirmed the patients complete recovery and no significant defects in his hearing or balance. We suggest multidisciplinary teamwork as a proper approach in the treatment of such injuries.

Craniocerebral Trauma↗

PubMed1999

Ubiquitous expression and embryonic requirement for RNA polymerase II coactivator subunit Srb7 in mice.

Mammalian RNA polymerase II complexes and coactivators containing homologs of yeast Srb/Med proteins have been isolated recently from tissue culture cells. The yeast Srb/Med complex is involved in global gene expression and is essential, but it is not yet known if its mammalian counterparts are broadly expressed in tissues or if they are essential. We have isolated the murine gene encoding Srb7, an Srb/Med complex protein whose sequence and function is highly conserved between yeast and humans. The mouse Srb7 gene is single copy, and Northern analysis showed that it is expressed in all tissues examined. Disruption of the gene in embryonic stem cells revealed that it is essential for cell viability and murine embryonic development. These results, together with evidence that murine Srb7 is associated exclusively with high molecular weight forms of RNA polymerase II in extracts, suggest that Srb7-containing polymerase complexes occur in most tissues and have essential roles in expression of protein coding genes.

Amino Acid Sequence↗

PubMed1999

Enzymatic and functional correction along with long-term enzyme secretion from transduced bone marrow hematopoietic stem/progenitor and stromal cells derived from patients with Fabry disease.

Fabry disease is a lysosomal storage disorder that is due to a deficiency in alpha-galactosidase A (alpha-gal A). Previously we have shown that a recombinant retrovirus synthesized for the transfer of the human alpha-gal A coding sequence was able to engineer enzymatic correction of the hydrolase deficiency in fibroblasts and lymphoblasts from Fabry patients. The corrected cells secreted alpha-gal A that was taken up and utilized by uncorrected bystander cells, thus demonstrating metabolic cooperativity. In separate experiments we used transduced murine bone marrow cells and successfully tested and quantitated this phenomenon in vivo. In the present studies, which were designed to bring this therapeutic approach closer to clinical utility, we establish that cells originating from the bone marrow of numerous Fabry patients and normal volunteers can be effectively transduced and that these target cells demonstrate metabolic cooperativity. Both isolated CD34+-enriched cells and long-term bone marrow culture cells, including nonadherent hematopoietic cells and adherent stromal cells, were transduced. The transferred gene generates increased intracellular alpha-gal A enzyme activity in these cells. Further, it causes functional correction of lipid accumulation and provides for long-term alpha-gal A secretion. Collectively, these results indicate that a multifaceted gene transfer approach to bone marrow cells may be of therapeutic benefit for patients with Fabry disease.

Animals↗

PubMed1999

Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila.

CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro, making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro, but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.

Animals↗

PubMed1998

Prediction of outcome in patients with missile craniocerebral injuries during the Croatian War.

The factors assumed to exert an influence on the outcomes of 176 patients who sustained head injuries through projectiles during the Croatian War were evaluated. The type of projectile, wound age, retained foreign bodies, and patient sex and age had no significant influence on outcome. Patients with a Glasgow Coma Scale score of 3 to 5 had 7.3 times higher relative risk of poor outcome than those with a score of 6 to 15. Patients with penetrating injuries (47%), with a projectile's path traversing both hemispheres (45.5%), and with intracranial hematomas (49%) had significantly poorer outcomes than patients without such lesions. Infections were more common in patients with retained foreign bodies in wounds that were older than 48 hours (43%) and in patients with cerebrospinal fluid fistulas (50%). In assessing the outcomes of missile head injuries in wartime, the Glasgow Coma Scale score, type of head wound, site of skull penetration, endocranial projectile path, intracranial hematomas, and complications, especially infectious, represent reliable predictors of outcome.

Adult↗

PubMed1997

UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior.

The end-to-end association of chromosomes through their telomeres has been observed in normal cells of certain organisms, as well as in senescent and tumor cells. The molecular mechanisms underlying this phenomenon are currently unknown. We show here that five independent mutant alleles in the Drosophila UbcD1 gene cause frequent telomere-telomere attachments during both mitosis and male meiosis that are not seen in wild type. These telomeric associations involve all the telomeres of the D. melanogaster chromosome complement, albeit with different frequencies. The pattern of telomeric associations observed in UbcD1 mutants suggests strongly that the interphase chromosomes of wild-type larval brain cells maintain a Rab1 orientation within the nucleus, with the telomeres and centromeres segregated to opposite sides of the nucleus. The UbcD1 gene encodes a class I ubiquitin-conjugating (E2) enzyme. This indicates that ubiquitin-mediated proteolysis is normally needed to ensure proper telomere behavior during Drosophila cell division. We therefore suggest that at least one of the targets of UbcD1 ubiquitination is a telomere-associated polypeptide that may help maintain proper chromosomal orientation during interphase.

Animals↗

PubMed1997

Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins.

MADS domain (for MCM1, AG, DEFA and SRF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled-coil structure suggests a role in establishing a protein-protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C-terminal region of AG in a yeast two-hybrid selection, 156 clones that encode potential AG-interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.

AGAMOUS Protein, Arabidopsis↗

PubMed1996

Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.

Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients, created as a hematologic delivery test system, were transduced. As with the fibroblasts, transduced patient B cell lines demonstrated both endogenous enzyme correction and a small amount of secretion together with uptake by uncorrected cells. These studies demonstrate that endogenous metabolic correction in transduced cells, combined with secretion, may provide a continuous source of corrective material in trans to unmodified patient bystander cells (metabolic cooperativity).

3T3 Cells↗

PubMed1996

A 1.5 kb repeat sequence flanks the suppressor of forked gene at the euchromatin-heterochromatin boundary of the Drosophila melanogaster X chromosome.

A 1.5 kilobasepair repeated DNA sequence is duplicated in direct orientation so as to flank the suppressor of forked gene in the euchromatin-heterochromatin transition region on the X chromosome of Drosophila melanogaster. These two copies are almost identical, but DNA blotting, analysis of cloned sequences and database searches show that elsewhere in the genome, homologous sequences are poorly conserved. They are often associated with other repeats, suggesting that they may belong to a scrambled and clustered middle repetitive DNA family. The sequences do not appear to be related to transposable elements and their location in different strains is conserved. In situ hybridization to metaphase chromosomes shows that homologous sequences are concentrated in the pericentric regions of the autosomes and the X chromosome. The sequences are not significantly under-represented in DNA from polytene tissue and must lie in the replicated regions of polytene chromosomes. The almost perfect conservation of the two repeats around suppressor of forked in D. melanogaster suggests they arose by duplication or gene conversion. Suppression of recombination in this chromosomal region presumably allows this unusual organization to be stably maintained. In the X-ray induced allele, suppressor of forked-L26, the sequence between the repeats, including the gene, and one copy of the repeat have been deleted.

Animals↗

PubMed1996

DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.

Amino Acid Sequence↗

PubMed1996

Functional domains of the floral regulator AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative mutations.

The Arabidopsis MADS box gene AGAMOUS (AG) controls reproductive organ identity and floral meristem determinacy. The AG protein binds in vitro to DNA sequences similar to the targets of known MADS domain transcription factors. Whereas most plant MADS domain proteins begin with the MADS domain, AG and its orthologs contain a region N-terminal to the MADS domain. All plant MADS domain proteins share another region with moderate sequence similarity called the K domain. Neither the region (I region) that lies between the MADS and K domains nor the C-terminal region is conserved. We show here that the AG MADS domain and the I region are necessary and sufficient for DNA binding in vitro and that AG binds to DNA as a dimer. To investigate the in vivo function of the regions of AG not required for in vitro DNA binding, we introduced several AG constructs into wild-type plants and characterized their floral phenotypes. We show that transgenic Arabidopsis plants with a 35S-AG construct encoding an AG protein lacking the N-terminal region produced apetala 2 (ap2)-like flowers similar to those ectopically expressing AG proteins retaining the N-terminal region. This result suggests that the N-terminal region is not required to produce the ap2-like phenotype. In addition, transformants with a 35S-AG construct encoding an AG protein lacking the C-terminal region produced ag-like flowers, indicating that this truncated AG protein inhibits normal AG function. Finally, transformants with a 35S-AG construct encoding an AG protein lacking both K and C regions produced flowers with more stamens and carpels. The phenotypes of the AG transformants demonstrate that both the K domain and the C-terminal region have important and distinct in vivo functions. We discuss possible mechanisms through which AG may regulate downstream genes.

Arabidopsis↗

PubMed1995

The Arabidopsis MADS-box gene AGL3 is widely expressed and encodes a sequence-specific DNA-binding protein.

The Arabidopsis AGL3 gene was previously identified on the basis of sequence similarity to the floral homeotic gene AGAMOUS (AG), which encodes a protein with a conserved MADS domain that is also found in human and yeast transcription factors (SRF and MCM1, respectively). Analysis of newly isolated full-length cDNA clones as well as genomic clones indicates that AGL3 is indeed a MADS-box gene with a general intron-exon structure similar to other plant MADS-box genes. However, unlike the others, which are expressed specifically in flowers, AGL3 is expressed in all above-ground vegetative organs, as well as in flowers, but not in roots. Furthermore, since AGL3 is MADS-domain protein, it is likely that it is also a DNA-binding protein regulating transcription. To characterize AGL3 as a DNA-binding protein in vitro, we expressed the AGL3 protein in Escherichia coli, and characterized its DNA-binding properties. We show that AGL3 binds to sequences which resemble the target sequences of SRF and MCM1, and have determined the consensus sequence to which AGL3 binds using random oligonucleotides. These results suggest that AGL3 is a widely distributed DNA-binding protein, which may be involved the transcriptional regulation of genes in many cells.

Amino Acid Sequence↗