Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoidtet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.

Despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. We recovered ribosomal DNA segments from a single human chromosome 21 using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering ∼0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies, several palindromic structures and potential errors in the previous reference sequence. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Approximately 60% of the 45S variants were confirmed in independent whole-genome or RNA-seq data, with 47 of these further observed in mature 18S/28S rRNA sequences. TAR cloning and long-read sequencing enabled the accurate reconstruction of multiple rDNA units and a new, high-quality 44 838 bp rDNA reference sequence, which we have annotated with variants detected from chromosome 21 of a single individual. The large number of variants observed reveal heterogeneity in human rDNA, opening up the possibility of corresponding variations in ribosome dynamics.

Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoidtetO-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoidtetO-HAC that has been formed from a ~50 kb synthetic alphoidtetO-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoidtetO-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo HAC formation in human cells.

It is well documented that mutations in the MYOCILINGlaucoma is a leading cause of blindness in the world. Primary open-angle glaucoma is the most common form of glaucoma. It will affect more than 60 million and blind about 4.5 million people worldwide by the year 2010 (62). It is now well established that a genetic component may contribute to glaucoma, and several glaucoma-associated genes have been identified. The first-identified and the most-studied gene is MYOCILIN (MYOC), which is highly expressed in and secreted by the trabecular meshwork (1,19,72,75,77,78), one of the key components of the eye aqueous humor outflow system. Dominant mutations in MYOC may lead to juvenile openangle glaucoma and are found in 3 to 4% of patients with primary open-angle glaucoma (18,19). The encoded protein, myocilin, belongs to a family of glycosylated proteins containing a C-terminal olfactomedin domain (57,90,91). Olfactomedin domain was originally identified in a glycoprotein isolated from the olfactory epithelium of frogs (71) and subsequently was found in a group of proteins forming a family of olfactomedin domaincontaining proteins. The family of olfactomedin domain-containing proteins includes both secreted and membrane-bound proteins that each exhibit a characteristic distribution in different tissues (4,10,27,30,44,51,58,78,79). Most of the glaucomacausing mutations in myocilin are located in the olfactomedin domain (1,2,18,19,24,72).Besides the trabecular meshwork and sclera (1, 77), high levels of MYOC were observed in the ciliary body (1, 13, 39), iris (89), retinal pigment epithelium/choroid (78, 88), and optic nerve (74). Low levels of MYOC expression were detected in several nonocular tissues (75). Myocilin, being a secreted protein, was also found in the aqueous humor, an intraocular fluid responsible for the supply of nutrients and for removal of metabolites from the avascular tissues of the eye anterior segment (63, 65). Some mutations in the MYOC gene lead to the inhibition of mutated myocilin secretion. Secretion of wildtype myocilin also can be reduced or blocked in the presence of mutated myocilin (22,36,52). It has been suggested that the intracellular accumulation of myocilin aggregates is deleterious to the trabecular meshwork cells, resulting in the deterioration of their function and subsequent elevation of intraocular pressure (IOP) (38,49).Although the MYOC gene has been studied for more than 10 years, the functions of myocilin protein are still not well understood (19,75). Biochemical data indicate that myocilin may interact with several intracellular and extracellular matrix proteins (16,37,48,61,73,78), although the biological significance of such interactions is not clear. The absence of openangle glaucoma in an elderly woman homozygous for the Arg46Stop mutation (45), as well as the absence of glaucoma in people hemizygous for MYOC (87), suggests that the loss of functional myocilin is not critical for the development of glaucoma or for normal eye functioning. These observations are supporte...

Whole-chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a distinguishing feature of most cancer types. CIN is generally considered to drive tumorigenesis, but a threshold level exists whereby further increases in CIN frequency in fact hinder tumor growth. While this attribute is appealing for therapeutic exploitation, drugs that increase CIN beyond this therapeutic threshold are currently limited. In our previous work, we developed a quantitative assay for measuring CIN based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Here, we used this assay to rank 62 different anticancer drugs with respect to their effects on chromosome transmission fidelity. Drugs with various mechanisms of action such as antimicrotubule activity, histone deacetylase (HDAC) inhibition, mitotic checkpoint inhibition, and targeting of DNA replication and damage responses were included in the analysis. Ranking of the drugs based on their ability to induce HAC loss revealed that paclitaxel, gemcitabine, dactylolide, LMP400, talazoparib, olaparib, peloruside A, GW843682, VX-680, and cisplatin were the top ten drugs demonstrating HAC loss at a high frequency. Therefore, identification of currently used compounds that greatly increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target and leverage the CIN phenotype in cancer cells.