Evidence for positive and negative regulation of the Hox-3.1 gene.

The region-specific patterns of expression of mouse homeobox genes are considered important for establishing the embryonic body plan. A 5-kilobase (kb) DNA fragment from the Hox-3.1 locus that is sufficient to confer region-specific expression to a beta-galactosidase reporter gene in transgenic mouse embryos has been defined. The observed reporter gene expression pattern closely parallels endogenous Hox-3.1 expression in 8- to 9.5-day postcoitum (p.c.) embryos. At 10.5 days p.c. and later, the pattern of beta-galactosidase activity diverges from the Hox-3.1 pattern, and an inappropriately high level of reporter gene expression is observed in posterior spinal ganglia. Inclusion of an additional 2 kb of upstream sequences is sufficient to suppress this aberrant expression in the developing spinal ganglia. Together, these results show that the control of early Hox-3.1 expression is complex, involving at least one positively acting and one negatively acting element.     

Targeted replacement of the homeobox gene Hox-3.1 by the Escherichia coli lacZ in mouse chimeric embryos.

Through gene targeting based upon homologous recombination in embryonic stem cells, a chosen gene can be inactivated and eventually a strain of mutant mice created. We have devised a procedure to specifically replace a targeted gene by another gene. A murine homeobox gene was disrupted at high frequency in embryonic stem cells by its replacement with Escherichia coli lacZ. Injection of such stem cells into blastocysts yielded chimeric embryos in which beta-galactosidase activity was driven by the Hox-3.1 promoter. This technique will allow the visual assessment at the cellular level of gene inactivation effects in transgenic mice.     

Structure and expression of Hox-2.2, a murine homeobox-containing gene.

The Hox-2.2 gene is one of a cluster of homeobox-containing genes on mouse chromosome 11. A cDNA clone containing the Hox-2.2 homeobox has been isolated from an adult spinal cord library. Our analysis of the Hox-2.2 cDNA and genomic clones indicates that there are at least two oxons and one intron. The largest open reading frame includes the homeobox and codes for a 224 amino acid protein of molecular weight 25,312. Comparisons of the predicted Hox-2.2 protein with other homeodomain-containing proteins revealed four regions of sequence similiarity: an N-terminal octapeptide, a hexapeptide upstream of the homeodomain, the homeodomain, and a glutamic acid-rich region at the C terminus. Possible functions of these regions are discussed. The Hox-2.2 gene is expressed in 13.5-day embryos in the developing hindbrain and spinal cord. The expression patterns of Hox-2.2 and Hox-2.1 in 13.5-day embryos are compared.     

Murine Hox-1.11 homeobox gene structure and expression.

The Hox-1.11 gene encodes a protein 372 amino acid residues long that contains a conserved pentapeptide, a homeodomain, and an acidic region. The amino acid sequence of the homeodomain of Hox-1.11 is identical to that of Hox-2.8, and the N-terminal and C-terminal regions of Hox-1.11 are similar to those of human HOX2H, which is the equivalent of murine Hox-2.8. The Hox-1.11 gene was shown to reside on murine chromosome 6, which contains the Hox-1 cluster of homeobox genes. One species of Hox-1.11 poly(A)+ RNA approximately 1.7 kb long was detected in mouse embryos, which is most abundant in 12-day-old embryos and progressively decreases during further embryonic development. The most anterior expression of Hox-1.11 poly(A)+ RNA in 12- to 14-day-old mouse embryos was shown by in situ hybridization to be in the mid and posterior hindbrain. Hox-1.11 poly(A)+ RNA also is expressed in the VII and VIII cranial ganglia, spinal cord, spinal ganglia, larynx, lungs, vertebrae, sternum, and intestine.     

Establishment of a human carcinoembryonic antigen (CEA)-producing cell line in a protein-free chemically defined medium

Summary To examine whether a human carcinoembryonic antigen (CEA)-producing cell can proliferate and sythesize CEA in vitro in culture without protein supplements, long-term cultivation of such cells was carried out in a protein-free chemically defined medium. Using stepwise decreases in fetal bovine serum concentration, continuous growth of the cells was established in a protein-free am's F-12 medium. The cells, designated as HLC-Yl, have been propagated in this medium for 3 years. The population doubling time of the cells is about 52 h. Addition of the serum stimulated the cell growth (population doubling time = 27 h). Saturation density was not increased by the addition of serum. The cells grown in a protein-free F-12 secrete large amounts of CEA (65.4±2.6 ng/10⁶ cells/24 h). Addition of serum did not stimulate the production of CEA. The cells produced tumours when inoculated into athymic nude mice. The mice bearing the tumour showed high serum CEA levels, and CEA was demonstrated in the tumour tissue by the immunoperoxidase method. The present study suggests that cells grown in a protein-free medium do not require serum components for their growth or CEA synthesis and provide an excellent model for better understanding the growth and production of CEA in human lung cancer cells.Abbreviations FBS fetal bovine serum - CEA carcinoembryonic antigen - PAP peroxidase anti-peroxidase - DAB Diaminobenzidine - PAS periodic acid-Schiff - PBS phosphate buffered saline - TEM transmission electron microscopy - SEM scanning electron microscopy     

Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line.

DNA from the human neuroblastoma cell line SK-N-SH is capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. Using genetic selection with the Escherichia coli sup F gene, we have isolated human sequences from mouse cells responsible for the oncogenic transformation. These sequences are present in all human DNAs surveyed and no gross rearrangements of these sequences are found in SK-N-SH cells. Although clearly distinct from two other human transforming genes present in bladder, lung, and colon carcinoma cell lines, all three transforming gene sequences may be related members of the ras gene family.     

Repression mediates cell-type-specific expression of the rat growth hormone gene.

A plasmid containing 1.8 kilobase pairs of rat growth hormone (rGH) promoter and upstream flanking sequences fused to the bacterial gene for chloramphenicol acetyltransferase (CAT) was transiently introduced into pituitary, fibroblast, and kidney cell lines. Significant CAT activity was detectable only in the pituitary cell lines, demonstrating that this relatively large fragment directs strongly cell-type-specific expression. However, plasmids containing only 200-300 bases of rGH promoter and flanking sequences directed expression of CAT in all three cell types, suggesting that upstream sequences directly repress the activity of a minimal rGH promoter in nonpituitary cell types. S1 nuclease analysis showed that the RNA synthesis directed by one of the short rGH promoter fragments in fibroblasts initiated from the site used by the natural promoter in pituitary cells. Insertion of rGH upstream sequences in their natural orientation upstream of the mouse metallothionein I promoter caused a decrease in its activity in fibroblasts by a factor of 4, but there was a 2.5-fold increase in its activity in pituitary cells. Insertion of the rGH fragment upstream of the thymidine kinase promoter in either orientation lowered its activity in both fibroblasts and pituitary cells. Thus, the negatively acting rGH flanking sequences can act on a heterologous promoter and have at least some of the properties of positively acting enhancers.     

Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line

The bcr-abl fusion gene is the molecular counterpart of the Philadelphia chromosome (Ph1) and is directly involved in the pathogenesis of Ph1+ leukemia. Inhibition of bcr-abl gene expression may have profound effects on the cell biology of Ph1+ cells, as recent experiments with antisense oligonucleotides have shown. In this study we have designed and synthesized a unique ribozyme that is directed against bcr-abl mRNA. The ribozyme cleaved bcr-abl mRNA in a cell-free in vitro system. A DNA-RNA hybrid ribozyme was then incorporated into a liposome vector and transfected into EM-2 cells, a cell line derived from a patient with blast crisis of chronic myelogenous leukemia. The ribozyme decreased levels of detectable bcr-abl mRNA in these cells, inhibited expression of the bcr-abl gene product, p210bcr-abl, and inhibited cell growth. This anti-bcr-abl ribozyme may be a useful tool to study the cell biology of Ph1+ leukemia and may ultimately have therapeutic potential in treating patients with Ph1 leukemias.     

Regulated expression of an immunoglobulin kappa gene introduced into a mouse lymphoid cell line.

We have introduced a functionally rearranged murine kappa light chain immunoglobulin (Ig) gene into an Abelson murine leukemia virus-transformed lymphoid cell line. Plasmid pSV2gpt-kappa 41, containing the kappa light chain gene from the myeloma MOPC41 and the selectable marker gene gpt, was introduced into 81A-2 cells by the calcium phosphate coprecipitation technique. Cells expressing the gpt gene were selected by growth in medium containing mycophenolic acid. One transfected cell line, kappa-2, was shown to make kappa mRNA and polypeptide chains and to assemble the kappa chain product with gamma 2b heavy chains to form an apparently complete IgG2b. When bacterial lipopolysaccharide was added to the growth medium, levels of kappa mRNA and polypeptide increased, showing regulated expression of the introduced kappa gene.