Structure and Function of Mammalian Centromere Section, LBC

Building 49, Room 4A56
301-496-7948

Summary of Research:

Our work can be divided into two interrelated parts. We are studying the general organization of centromeric regions of human chromosomes. The major focus of the research being conducted in our laboratory is to determine minimal structural requirements for functional centromere and to generate a portable human artificial chromosome (HAC) for gene delivery.  We are also working on isolation and characterization of human disease genes. Ongoing work is presently focused on two major areas of research.

The Organization of Human Centromere

The mammalian centromere is a specialized region of the chromosome, which controls proper chromosome segregation. Structural studies of mammalian centromeres have shown that they contain long stretches of tandemly repeated DNA sequences. These regions represent approximately 15% of human genome.  Despite their importance, centromeres remain poorly understood and even a new released human genome sequence does not contain information on centromeric regions. Previously it was thought that this fraction of genome lacks genes and represents a junk DNA. Recent identification of functional copies of genes within centromeric regions of plant chromosomes raises the question that human centromeres spanning several megabases may be organized similarly.  The main reason why centromeres remain poorly understood is that long stretches of tandemly repeated centromere-specific DNA sequences can not be cloned by a standard technique and they are unstable during propagation in both bacterial and yeast hosts.  We developed a novel approach for isolating of centromeric regions based on specific targeting of centromeric DNA and the following rescue of the targeting region as a set of large yeast artificial chromosomes (YACs). This new cloning strategy was applied for studying several centromeric regions including a centromeric region of a mini-chromosome containing ~5 Mb of the human chromosome Y.  William Brown and co-authors (University of Oxford) generated this mini-chromosome by two rounds of telomere-directed chromosome breakage leading to a loss of sequences from both arms of the chromosome.  Despite the small size and loss of a significant part of centromeric repeats (only 140 kb of alphoid DNA was left), the mini-chromosome segregates accurately in mitosis, suggesting that this block of alphoid DNA alone or along with the short arm flanking sequence is sufficient for a centromere function.  As a first step to construct Human Artificial Chromosome (HAC) the centromeric region of the mini-chromosome was TAR rescued in yeast as a circular YAC and its complete sequence was determined.  For further functional analysis the YAC was retrofitted into a YAC/BAC with a mammalian selectable marker.  When transfected into human cells, this circular DNA construct is stably maintained at 1-2 copies per cell. We are in progress to investigate the HAC as a vector for megabase-size genes and as a model for studying a human kinetohore.

The Organization and Expression of Human Diseases Genes

The mammalian gene function analysis is impeded by the lack of a convenient expression system.  Attempts to express human genes using cDNA expression vectors frequently result in cell cycle arrest as a result of uncontrolled expression of the genes in transfected cells. It is obvious that a genomic copy of the genes with all regulatory elements (including potential regulatory elements in intron regions) instead of surrogate cDNA constructs would be preferential for gene expression studies. However,  routine large insert size cloning techniques (BAC and YAC) produce clones with random genomic fragments  and the gene of interest is either available only as a set of overlapping genomic fragments that form a contig or available as a large genomic fragment carrying other genes.  A new approach referred to as TAR (Transformation-Associated Recombination) cloning has recently emerged, which allows entire genes and large chromosome regions to be specifically and accurately isolated from total genomic DNA. This nonenzymatic procedure for DNA cloning is based on in vivo recombination in the yeast S. cerevisiae, an organism that exhibits a high level of intermolecular recombination between homologous DNAs during transformation.

Over the last two years we successfully applied the new technique for isolation of entire copies of several human genes including breast cancer genes, BRCA1 and BRCA2, the metastasis-suppressor gene KAI1, the telomerase reverse transcriptase gene hTERT and a paternally expressed gene Ubiq. We are in progress to investigate regulation of expression of these genes using a recently developed Human Artificial Chromosome (HAC) as a gene delivery system.

Vladimir Larionov, Ph.D., Acting Head
Natalay Kouprina
Vladimir Noskov
Maxim Koriabine