Hybridization of nucleic acids to surface-tethered oligonucleotide probes has numerous potential applications in genome mapping and DNA sequence analysis. In this article, we describe a simple standard protocol for routine preparation of terminal amine-derivatized 9-mer oligonucleotide arrays on ordinary microscope slides and hybridization conditions with DNA target strands of up to several hundred bases in length with good discrimination against mismatches. Additional linker arms separating the glass surface from the probe sequence are not necessary. The technique described here offers a powerful tool for the detection of specific genetic mutations.

Microfabricated devices containing arrays of nucleic acid hybridization sites, known as genosensors, are being developed for a variety of uses in genomic analysis. A great deal of the overall genosensor development effort involves optimization of experimental conditions in the actual use of genosensors. Here we describe a "low-tech" form of genosensor technology, involving arrays of oligonucleotides on glass microscope slides, which can be used to define optimal operating conditions and to develop applications of hybridization arrays in genome mapping and sequencing. In addition, we describe a porous silicon genosensor, which can be operated in a flowthrough mode, and discuss its advantages over current flat-surface designs. Porous silicon genosensors containing arrays of DNA fragments offer several unique capabilities in genome analysis.

The eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs). CDK4 and CDK6, which are activated by D-type cyclins during the G 1 phase of the cell cycle, are thought to be responsible for phosphorylation of the retinoblastoma gene product (pRb). The tumor suppressor p16INK4A inhibits phosphorylation of pRb by CDK4 and CDK6 and can thereby block cell cycle progression at the G 1 /S boundary. Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II by general transcription factor TFIIH is believed to be an important regulatory event in transcription. TFIIH contains a CDK7 kinase subunit and phosphorylates the CTD. We have previously shown that p16INK4A inhibits phosphorylation of the CTD by TFIIH. Here we report that the ability of p16INK4A to inhibit CDK7-CTD kinase contributes to the capacity to induce cell cycle arrest. These results suggest that p16INK4A may regulate cell cycle progression by inhibiting not only CDK4-pRb kinase activity but also by modulating CDK7-CTD kinase activity. Regulation of CDK7-CTD kinase activity by p16INK4A thus may represent an alternative pathway for controlling cell cycle progression.Cyclin-dependent kinases (CDKs) regulate cell cycle progression (references 13, 21, and 28) and references therein). CDK4 and CDK6 are activated by D-type cyclins and participate in controlling the G 1 -to-S phase transition by phosphorylating the retinoblastoma gene product (pRb). Phosphorylation of pRb induces remodeling of transcriptional repressor complexes at pRb-regulated genes and causes the release of transcription factors such as E2F. Free E2F can then activate the transcription of genes required for entering S phase (36,41). p16 INK4A is a tumor suppressor gene product which binds CDK4 and inhibits CDK4-mediated phosphorylation of pRb (27). Overexpression of p16INK4A can block cell cycle progression through the G 1 -to-S phase boundary in a pRB-dependent manner (16,19). Many p16 INK4A mutants identified from human tumors have been shown to have defects in this activity (15,16,19,20,22,31). These data suggest that the CDK4-inhibitory activity of p16 INK4A is involved in regulating cell cycle progression through the G 1 /S boundary.Koh et al. have described an interesting phenotype associated with a p16INK4A mutant, G101W, that was originally identified in a familial melanoma kindred (14, 16). The G101W mutant was defective in inhibiting CDK4, although overexpression of the G101W mutant in an osteosarcoma cell line provoked cell cycle arrest at G 1 . In this mutant, the CDK4-pRb kinase-inhibitory activity of p16 INK4A apparently does not correlate with the ability to induce cell cycle arrest in G 1 when overexpressed. These results raise the possibility that an additional biochemical activity of p16INK4A might contribute to the ability to arrest cell cycle progression. (15,16,19,20,22,31,38,39). These data suggest that the ability to inhibit pRb kinase activity may not be the sole determinant of the tumor suppressor activity of p16 INK4A ....

Several studies have demonstrated that chronic treatment with nicotine elicits an increase in the number of brain nicotinic receptors. To determine whether this effect is elicited by other nicotinic agonists found in tobacco, the effects of chronic infusion with nicotine on brain nicotinic receptors were compared with those after anabasine and lobeline. C57BL/6 mice were infused with saline or equimolar doses (18.5 mumol/kg/h) of nicotine, anabasine, or lobeline for 8 days. Nicotinic receptors, quantified by the binding of [3H]nicotine and [125I]iodo-alpha-bungarotoxin (alpha-[125I]BTX), and muscarinic receptors, quantified by the binding of [3H]quinuclidinyl benzilate ([3H]QNB), were then assayed in eight brain regions. An increase in [3H]nicotine binding was observed in all regions except cerebellum following chronic infusion with nicotine and anabasine, whereas lobeline did not alter the number or affinity of these binding sites. This increase was due to changes in Bmax and not in the affinity of the receptor for the ligand (KD). A slight increase in alpha-[125I]BTX binding was observed in cortex following chronic anabasine infusion. [3H]QNB binding sites were largely unaltered following chronic infusion with any of the nicotinic analogs. The levels of the agonists in the brain were also determined after chronic treatment, and the amounts of lobeline and anabasine were found to be higher than that of nicotine. Thus, the failure of lobeline to elicit changes in nicotine binding is not due to reduced brain concentrations.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects has been described by other workers, however, relatively few details of the in vitro growth of the cells have been published. Here we describe the release of cells from adult human articular cartilage and their growth characteristics in vitro.Cultures were successfully established from 29 of 30 biopsies taken from patients aged 20-72 year. No significant relationship was found between donor age and initial cell yield following cartilage digest, however, the time to primary confluence increased in direct proportion to age. Thereafter the kinetics of cell proliferation was independent of donor age.The proportion of apoptotic or necrotic cells in the cartilage digest was low and increased with time in culture only in those cells which remained non-adherent. Conversely, entry into cell cycle was restricted to those cells which had become adherent.These results illustrate that previously reported techniques for isolating and culturing chondrocytes are reproducible, that adherent chondrocytes have considerable proliferative potential, and that concern about cell growth and viability need not, in itself, limit the clinical application of ACI to younger patients.