Active Role of a Human Genomic Insert in Replication of a Yeast Artificial Chromosome

ABSTRACT Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

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Physical mapping of the human T-cell receptor beta gene complex, using yeast artificial chromosomes

Immunogenetics ◽

10.1007/bf00163990 ◽

1995 ◽

Vol 41 (6) ◽

pp. 337-342 ◽

Cited By ~ 4

Author(s):

Yasmeen Hashim ◽

Ioannis Ragoussis ◽

Lyndal Kearney ◽

Sabrina Tosi ◽

Alex K. So

Keyword(s):

T Cell Receptor ◽

Physical Mapping ◽

Cell Receptor ◽

Gene Complex ◽

Artificial Chromosomes ◽

Human T Cell ◽

T Cell Receptor Beta ◽

Yeast Artificial Chromosomes ◽

Beta Gene ◽

Receptor Beta

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An Artificially Constructed De Novo Human Chromosome Behaves Almost Identically to Its Natural Counterpart during Metaphase and Anaphase in Living Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00355-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7682-7695 ◽

Cited By ~ 16

Author(s):

Tomohiro Tsuduki ◽

Megumi Nakano ◽

Nao Yasuoka ◽

Saeko Yamazaki ◽

Teruaki Okada ◽

...

Keyword(s):

Yeast Artificial Chromosome ◽

Fluorescent Protein ◽

De Novo ◽

Artificial Chromosome ◽

Time Lapse ◽

Living Cells ◽

Lac Repressor ◽

Host Chromosome ◽

Artificial Chromosomes ◽

Spindle Midzone

ABSTRACT Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing α-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input α-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

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The Human T cell Receptor Beta Variable (TRBV) Genes

Experimental and Clinical Immunogenetics ◽

10.1159/000019123 ◽

2000 ◽

Vol 17 (1) ◽

pp. 42-54 ◽

Cited By ~ 49

Author(s):

Géraldine Folch ◽

Marie-Paule Lefranc

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Human T Cell ◽

T Cell Receptor Beta ◽

Beta Variable ◽

Receptor Beta

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Detection of myc translocations in lymphoma cells by fluorescence in situ hybridization with yeast artificial chromosomes

Blood ◽

10.1182/blood.v85.8.2132.bloodjournal8582132 ◽

1995 ◽

Vol 85 (8) ◽

pp. 2132-2138 ◽

Cited By ~ 23

Author(s):

ML Veronese ◽

M Ohta ◽

J Finan ◽

PC Nowell ◽

CM Croce

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Yeast Artificial Chromosome ◽

Human Lymphocytes ◽

Artificial Chromosome ◽

Chromosome 8 ◽

Metaphase Chromosomes ◽

Artificial Chromosomes ◽

Myc Gene

Translocations involving chromosome 8 at band q24 and one of the Ig loci on chromosomes 14q32, 22q11, and 2p11 are the hallmark of Burkitt's lymphoma (BL). It has been previously observed that the exact localization of the breakpoints at chromosome 8q24 can vary significantly from patient to patient, scattering over a distance of more than 300 kb upstream of c-myc and about 300 kb downstream of c-myc. To generate probes for fluorescence in situ hybridization (FISH) that detect most c-myc translocations, we screened a yeast artificial chromosome (YAC) library from normal human lymphocytes by colony hybridization, using three markers surrounding the c-myc gene as probes. We obtained 10 YAC clones ranging in size between 500 and 200 kb. Two nonchimeric clones were used for FISH on several BL cell lines and patient samples with different breakpoints at 8q24. Our results show that the YAC clones detected translocations scattered along approximately 200 kb in both metaphase chromosomes and interphase nuclei. The sensitivity, rapidity, and feasibility in nondividing cells render FISH an important diagnostic tool. Furthermore, the use of large DNA fragments such as YACs greatly simplifies the detection of translocations with widely scattered breakpoints such as these seen in BL.

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Diversity and structure of human T-cell receptor beta-chain variable region genes.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.83.17.6598 ◽

1986 ◽

Vol 83 (17) ◽

pp. 6598-6602 ◽

Cited By ~ 100

Author(s):

P. Concannon ◽

L. A. Pickering ◽

P. Kung ◽

L. Hood

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Variable Region ◽

Cell Receptor ◽

Beta Chain ◽

Human T Cell ◽

T Cell Receptor Beta ◽

Variable Region Genes ◽

Receptor Beta

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Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.23.11381 ◽

1993 ◽

Vol 90 (23) ◽

pp. 11381-11385 ◽

Cited By ~ 88

Author(s):

K M Gaensler ◽

M Kitamura ◽

Y W Kan

Keyword(s):

Transgenic Mice ◽

Yeast Artificial Chromosome ◽

Germ Line ◽

Physiological Role ◽

Artificial Chromosome ◽

Chromatin Domain ◽

Globin Genes ◽

Beta Globin ◽

Cis Acting ◽

Artificial Chromosomes

Sequential expression of the genes of the human beta-globin locus requires the formation of an erythroid-specific chromatin domain spanning > 200 kb. Regulation of this gene family involves both local interactions with proximal cis-acting sequences and long-range interactions with control elements upstream of the locus. To make it possible to analyze the interactions of cis-acting sequences of the human beta-globin locus in their normal spatial and sequence context, we characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire beta-globin locus. We have now successfully integrated the 150-kb YAC into the germ line of transgenic mice as a single unrearranged fragment that includes the locus control region, structural genes, and 30 kb of 3' flanking sequences present in the native locus. Expression of the transgenic human beta-globin locus is tissue- and developmental stage-specific and closely follows the pattern of expression of the endogenous mouse beta-globin locus. By using homology-directed recombination in yeast and methods for the purification and transfer of YACs into transgenic mice, it will now be feasible to study the physiological role of cis-acting sequences in specifying an erythroid-specific chromatin domain and directing expression of beta-globin genes during ontogeny.

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Arabidopsis YAC restriction mapping

Genome ◽

10.1139/g98-086 ◽

1998 ◽

Vol 41 (6) ◽

pp. 806-817

Author(s):

Shaun M Morroll ◽

Zoe A Wilson

Keyword(s):

Positional Cloning ◽

Floral Development ◽

Yeast Artificial Chromosome ◽

Artificial Chromosome ◽

Genomic Region ◽

Restriction Mapping ◽

Artificial Chromosomes ◽

Number Of Genes ◽

Yeast Artificial Chromosomes ◽

Large Genomic Region

The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (~27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region. The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used. This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterised genes involved in male gametogenesis (e.g., Ms1 and Ms37). The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterised floral development genes which lie in this region. This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.Key words: yeast artificial chromosome, YAC, Arabidopsis thaliana, partial restriction mapping, floral development.

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