Biomedical subjects

Izabela Makalowska

Publications and source records attributed to Izabela Makalowska.

9 recordsLinked to original sources

PubMed2004

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

Alternative Splicing↗

PubMed2003

Identification and characterization of mouse Rab32 by mRNA and protein expression analysis.

Rab proteins, a subfamily of the ras superfamily, are low molecular weight GTPases involved in the regulation of intracellular vesicular transport. Cloning of human RAB32 was recently described. Presently, we report the cloning and characterization of the mouse homologue of Rab32. We show that murine Rab32 exhibits a ubiquitous expression pattern, with tissue-specific variation in expression level. Three cell types with highly specialized organelles, melanocytes, platelets and mast cells, exhibit relatively high level of Rab32. We show that in murine amelanotic in vitro transformed melanocytes as well as in human amelanotic metastatic melanoma cell lines, the expression of Rab32 is markedly reduced or absent, in parallel with the loss of expression of two key enzymes for the production of melanin, tyrosinase and Tyrp1. Therefore, in both mouse and human systems, the expression of Rab32 correlates with the expression of genes involved in pigment production. However, in melanoma samples, amelanotic due to a mutation in the tyrosinase gene, the expression of Rab32 remains at levels comparable to those observed in pigmented melanoma samples. Finally, we observed co-localization of Rab32 and the melanosomal proteins, Tyrp1 and Dct, indicating an association of Rab32 with melanosomes. Based on these data, we propose the inclusion of Rab32 to the so-called melanocyte/platelet family of Rab proteins.

Animals↗

PubMed2003

Cloning and characterization of a novel gene, SHPRH, encoding a conserved putative protein with SNF2/helicase and PHD-finger domains from the 6q24 region.

Here we report the identification of a novel transcript containing SNF2, PHD-finger, RING-finger, helicase, and linker histone domains mapping to the q24 band region of human chromosome 6. These domains are characteristic of several DNA repair proteins, transcription factors, and helicases. We have cloned both human and mouse homologs of this novel gene using interexon PCR and RACE technologies. The human cDNA, termed SHPRH, is 6018 bp and codes for a putative protein of 1683 amino acids. The mouse cDNA, termed Shprh, is 7225 bp and codes for a putative protein of 1616 amino acids. The deduced amino acid sequences of the two proteins share 86% identity. Both genes are expressed ubiquitously, with a transcript size of approximately 7.5 kb. Mapping of this gene to 6q24, a region reported to contain a tumor suppressor locus, prompted us to evaluate SHPRH by mutation analysis in tumor cell lines. We have identified one truncating and three missense mutations, thus suggesting SHPRH as a possible candidate for the tumor suppressor gene.

Amino Acid Sequence↗

PubMed2003

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

Animals↗

PubMed2003

GALA, a database for genomic sequence alignments and annotations.

We have developed a relational database to contain whole genome sequence alignments between human and mouse with extensive annotations of the human sequence. Complex queries are supported on recorded features, both directly and on proximity among them. Searches can reveal a wide variety of relationships, such as finding all genes expressed in a designated tissue that have a highly conserved noncoding sequence 5' to the start site. Other examples are finding single nucleotide polymorphisms that occur in conserved noncoding regions upstream of genes and identifying CpG islands that overlap the 5' ends of divergently transcribed genes. The database is available online at http://globin.cse.psu.edu/ and http://bio.cse.psu.edu/.

5' Untranslated Regions↗

PubMed2002

Mutant deoxynucleotide carrier is associated with congenital microcephaly.

The disorder Amish microcephaly (MCPHA) is characterized by severe congenital microcephaly, elevated levels of alpha-ketoglutarate in the urine and premature death. The disorder is inherited in an autosomal recessive pattern and has been observed only in Old Order Amish families whose ancestors lived in Lancaster County, Pennsylvania. Here we show, by using a genealogy database and automated pedigree software, that 23 nuclear families affected with MCPHA are connected to a single ancestral couple. Through a whole-genome scan, fine mapping and haplotype analysis, we localized the gene affected in MCPHA to a region of 3 cM, or 2 Mb, on chromosome 17q25. We constructed a map of contiguous genomic clones spanning this region. One of the genes in this region, SLC25A19, which encodes a nuclear mitochondrial deoxynucleotide carrier (DNC), contains a substitution that segregates with the disease in affected individuals and alters an amino acid that is highly conserved in similar proteins. Functional analysis shows that the mutant DNC protein lacks the normal transport activity, implying that failed deoxynucleotide transport across the inner mitochondrial membrane causes MCPHA. Our data indicate that mitochondrial deoxynucleotide transport may be essential for prenatal brain growth.

Carrier Proteins↗

PubMed2002

Identification of six novel genes by experimental validation of GeneMachine predicted genes.

In silico gene identification from finished and unfinished human genome sequence has become critically important in many projects seeking to gain insights into the gene content of genomic regions implicated in diseases. To establish limitations and criteria for in silico gene identification, and to identify novel genes of potential relevance to human prostate cancer and melanoma, 3 Mb of chromosome 1 sequence have been analyzed using GeneMachine. This program is a software suite comprising of sequence similarity programs and four gene identification programs. A total of 49 potential transcripts were selected and 37 of them were selected for experimental validation. We verified 16 of the predicted genes by experimental analysis. The comparison of the predicted transcripts with their cloned forms helped to refine predicted gene models as well as to identify splice variants for several of them. Although sequences matching with ten of our verified genes have been recently deposited in the GenBank, six of them remain novel. Our studies support the feasibility of identifying novel genes from regions of interest using draft human genome sequence.

Chromosomes, Human, Pair 1↗

PubMed2002

The Histone Database.

Histone proteins are often noted for their high degree of sequence conservation. It is less often recognized that the histones are a heterogeneous protein family. Furthermore, several classes of non-histone proteins containing the histone fold motif exist. Novel histone and histone fold protein sequences continue to be added to public databases every year. The Histone Database (http://genome.nhgri.nih.gov/histones/) is a searchable, periodically updated collection of histone fold-containing sequences derived from sequence-similarity searches of public databases. Sequence sets are presented in redundant and non-redundant FASTA form, hotlinked to GenBank sequence files. Partial sequences are also now included in the database, which has considerably augmented its taxonomic coverage. Annotated alignments of full-length non-redundant sets of sequences are now available in both web-viewable (HTML) and downloadable (PDF) formats. The database also provides summaries of current information on solved histone fold structures, post-translational modifications of histones, and the human histone gene complement.

Amino Acid Motifs↗

PubMed2002

Physical and transcript map of the hereditary prostate cancer region at xq27.

We have recently mapped a locus for hereditary prostate cancer (termed HPCX) to the long arm of the X chromosome (Xq25-q27) through a genome-wide linkage study. Here we report the construction of an approximately 9-Mb sequence-ready bacterial clone contig map of Xq26.3-q27.3. The contig was constructed by screening BAC/PAC libraries with markers spaced at approximately 85-kb intervals. We identified overlapping clones by end-sequencing framework clones to generate 407 new sequence-tagged sites, followed by PCR verification of overlaps. Contig assembly was based on clone restriction fingerprinting and the landmark information. We identified a minimal overlap contig for genomic sequencing, which has yielded 7.7 Mb of finished sequence and 1.5 Mb of draft sequence. The transcriptional mapping effort localized 57 known and predicted genes by database searching, STS content mapping, and sequencing, followed by sequence annotation. These transcriptional units represent candidate genes for HPCX and multiple other hereditary diseases at Xq26.3-q27.3.

Chromosome Mapping↗