Larry L. Deaven

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.

ISOLATION OF A B-CELL-SPECIFIC PROMOTER FOR THE HUMAN CLASS II TRANSACTIVATOR

Abstract The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-γ) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-γ treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-γ-inducible expression of CIITA.

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5

B‐cell chronic lymphocytic leukemia (B‐CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2‐42), which corresponds to the minimal region shared by B‐CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc‐finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B‐CLL rearrangements.

The temporal structure of S phase

DNA synthesis in the S phase of V79 and CHO Chinese hamster cells was examined in detail using an automated system for selection and subculturing of mitotic cells and four different assays for DNA synthesis. Flow microfluorometric (FMF) analysis showed that the selected populations were highly synchronous with few noncycling cells. In CHO cells changes in mean and modal fluorescence in the FMF suggested that DNA content increased in a saltatory fashion with 10-20% of the DNA replicated in early S, 40% in mid S, and 40-50% in late S. Pulse labeling and acid precipitation revealed a repeatable pattern of fluctuations in the rate of isotope incorporation with the maximum rate occurring late in S both V79 and CHO. Autoradiography proved to be the best means of accurately determining the beginning of S phase. Cumulative labeling from mitosis to points in S exaggerated the differences in rate between early and late S, so that significant DNA synthesis in early S might easily be overlooked using this method. In CHO cells DNA-specific fluorescence by the Kissane and Robins assay supported the isotopic incorporation data and the FMF analyses by exhibiting a stepwise increase. In V79 cells, S phase lasts only 5 or 5.5 hr, and consequently the mid S and late S steps in fluorescence are compressed. In V79, greater than 80% of the increase in DNA-specific fluorescence occurred between 4.5 and 7 hr after mitotic selection. In both cell lines, fluorescence in early S phase frequently increased transiently to maximum and then decreased.

A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis

The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously.

A cDNA from the ovarian cancer critical region of deletion on chromosome 17p13.3

Chromosome 17p13.3 is frequently deleted in human ovarian carcinoma, and the 15 kb critical region of deletion may contain a tumor suppressor gene. A 2.3 kb cDNA has been identified which spans 17 kb of genomic DNA, including 8.1 kb within the critical region, and thus is a candidate tumor suppressor gene. This highly conserved gene has significant sequence similarity to a yeast gene of unknown function and to one of the yeast enzymes in the diphthamide synthetic pathway, DPH2, that has a role in global protein synthesis regulation. This gene, named DPH2L (diphthamide biosynthesis protein 2-like), is expressed in multiple tissues and stages of development.

Construction and characterization of partial digest DNA libraries made from flow-sorted human chromosome 16☆

In this report, we present the techniques used for the construction of chromosome-specific partial digest libraries from flow-sorted chromosomes and the characterization of two such libraries from human chromosome 16. These libraries were constructed to provide materials for use in the development of a high-resolution physical map of human chromosome 16, and as part of a distributive effort on the National Laboratory Gene Library Project. Libraries with 20-fold coverage were made in Charon-40 (LA16NL03) and in sCos-1 (LA16NC02) after chromosome 16 was sorted from a mouse-human monochromosomal hybrid cell line containing a single homologue of human chromosome 16. Both libraries are approximately 90% enriched for human chromosome 16, have low nonrecombinant backgrounds, and are highly representative for human chromosome-16 sequences. The cosmid library in particular has provided a valuable resource for the isolation of coding sequences, and in the ongoing development of a physical map of human chromosome 16.

Characterization of a human chromosome 8 cosmid library constructed from flow-sorted chromosomes

A cosmid library for human chromosome 8 has been constructed from flow-sorted chromosomes in the vector sCos-1. This library is 85% human and has been arrayed into 210 microtiter plates representing four genome equivalents. Cosmids have been isolated with 10 of 11 probes representing nine different loci from chromosome 8.

The gene for human chromogranin A (CgA) is located on chromosome 14.

Chromogranin A (CgA) is a protein that is present in most neuroendocrine tissues and is co-secreted with their resident hormones. We have assigned the CgA gene to human chromosome 14 by hybridization of a CgA cDNA probe cloned from a cDNA library of human medullary thyroid carcinoma cells to spots of individual human chromosomes flow-sorted onto nitrocellulose filters. Southern analysis of human genomic DNA with the same probe revealed only 1-3 restriction bands. These studies indicate that the CgA gene is probably single copy and not a member of a dispersed, multigene family. The CgA gene is not co-localized with the genes of any of the CgA-associated hormones.

Mapping of human lactate dehydrogenase-A, -B, and -C genes and their related sequences: the gene for LDHC is located with that for LDHA on chromosome 11

Human testis-specific lactate dehydrogenase-C (LDHC) gene-related sequences are located with the LDHA gene on chromosome 11. The LDHB gene is on chromosome 12. Chromosomes 1, 2, 4, 9, and 10 appear to contain LDHA gene-related sequences, whereas the X chromosome and chromosome 13 possess LDHB gene-related sequences.