The Saccharomyces cerevisiae kinetochore contains a cyclin-CDK complexing homologue, as identified by in vitro reconstitution.

We have developed methods to reconstitute the centromere DNA (CEN)-bound Saccharomyces cerevisiae kinetochore complex, CBF3, from isolated CBF3 components in vitro. This revealed that cooperation of at least three CBF3 components is imperatively required to form an activity that specifically binds to the centromere DNA in vitro. Two of the CBF3 proteins, Cbf3a and Cbf3b, that were used in the reconstitution were obtained from heterologous systems. In contrast, Cbf3c, the third CBF3 component known, had to be purified from S. cerevisiae to obtain a Cbf3c preparation that was competent to reconstitute the CBF3-CEN complex in combination with Cbf3a and Cbf3b. This led to the identification of a 29 kDa protein that co-purified with Cbf3c. The 29 kDa protein was shown to be a fourth component of CBF3 and therefore was named Cbf3d. Analysing the Cbf3d gene revealed that Cbf3d exhibits strong homology to p19SKP1, a human protein that is part of active cyclin A-CDK2 complexes. Therefore, Cbf3d is the only CBF3 protein that has a known homologue in higher eukaryotes and may provide the anchor that directs cell cycle-regulated proteins to the kinetochore.     

Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.      

A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein

This paper describes the construction of a DNA molecule containing a topologically stable structure that simulates a replication fork. This preformed DNA molecule is a circular duplex of 7.2 X 10(3) base pairs (M13mp6 DNA) from which arises, at a unique BamHI recognition site, a noncomplementary 5'-phosphoryl-terminated single strand of 237 nucleotides (SV40 DNA). This structure has two experimental attributes. 1) Templates for both leading and lagging strand synthesis exist as stable structures prior to any DNA synthesis. 2) DNA synthesis creates a cleavage site for the restriction endonuclease BamHI. Form I of T7 DNA polymerase, alone, catalyzes limited DNA synthesis at the preformed replication fork whereas Form II, alone, polymerizes less than 5 nucleotides. However, when T7 gene 4 protein is present, Form II of T7 DNA polymerase catalyzes rapid and extensive synthesis via a rolling circle mode. Kinetic analysis of this synthesis reveals that the fork moves at a rate of 300 bases/s at 30 degrees C. We conclude that the T7 gene 4 protein requires a single-stranded DNA binding site from which point it translocates to the replication fork where it functions as a helicase. The phage T4 DNA polymerase catalyzes DNA synthesis at this preformed replication fork in the presence of gene 4 protein, but the amount of DNA synthesized is less that 3% of the amount synthesized by the combination of Form II of T7 DNA polymerase and gene 4 protein. We conclude that T7 DNA polymerase and T7 gene 4 protein interact specifically during DNA synthesis at a replication fork.     

The dynamics of formation of a collagen-phosphophoryn conjugate in relation to the passage of the mineralization front in rat incisor dentin

Dentin and predentin matrices contain Type I collagen and phosphophoryns as major constituents. A collagen-phosphophoryn conjugate is also present in small amounts. This conjugate has been implicated in the deposition of mineral. Its formation has been followed in rat incisors. Rats were labeled for varied time intervals with [3H]proline, followed by a 2-h pulse of [3H] serine. The soluble alpha- and beta-phosphophoryns were extracted under conditions minimizing degradation. The tooth residue was CNBr-treated and the collagen CNBr peptides alpha 1(I)CB7 and alpha 1(I)CB8 were collected along with the solubilized conjugate fraction. Each component was purified and the specific activities in [3H] proline, [3H]hydroxyproline, [3H]serine, and [3H]phosphoserine were determined. The collagen and alpha-phosphophoryn accumulated proline label linearly at the same rate over the entire period of labeling. Entry of [3H]proline into the conjugate fraction was delayed by approximately 9-10 h and then the label accumulated also linearly at the same rate. [3H]Serine was present at a different but constant level in each fraction; the conjugate had the lowest activity. These data indicate an extracellular formation of the conjugate at the mineralization front from precursors which followed different secretory pathways.     

Bombesin and the C-terminal tetradecapeptide of gastrin-releasing peptide are growth factors for normal human bronchial epithelial cells

Bombesin and the C-terminal portion of gastrin-releasing peptide (GRP14-27) each increase clonal growth rate and colony-forming efficiency of normal human bronchial epithelial cells. These effects occur in the presence or absence of an optimal concentration (5 ng/ml) of epidermal growth factor (EGF). In contrast to EGF bombesin and GRP14-27 do not stimulate cell migration. Thus, bombesin and the C-terminal fragment of gastrin-releasing peptide represent a new class of peptides mitogenic for normal human epithelial cells.     

Human bronchial epithelial cells with integrated SV40 virus T antigen genes retain the ability to undergo squamous differentiation

Human bronchial epithelial cells transformed by either DNA virus infection (SV40 or Adenovirus 12-SV40 hybrid virus) or transfection with the SV40 large T antigen gene were studied for their ability to undergo squamous differentiation when exposed to 12-O-tetradecanoylphorbol-13 acetate (TPA), transforming growth factor-beta 1 (TGF-beta 1), or fetal bovine serum (FBS), agents that induce the squamous differentiation of normal human bronchial epithelial cells. Squamous differentiation occurred in all ten T-antigen-positive cell cultures when they were exposed to either FBS or TGF-beta 1, but none differentiated when exposed to TPA. From one cell line, designated BEAS-2B, two subclones were isolated, one of which was induced to undergo squamous differentiation by FBS, and a second that failed to undergo squamous differentiation and was mitogenically stimulated when exposed to serum. These phenotypically different subclones provide a new in vitro cellular system for delineating the mechanism(s) of human bronchial epithelial cell squamous differentiation in response to FBS or TGF-beta 1.     

Differential responses to growth factors by normal human mesothelial cultures from individual donors

A significant interindividual variation in the growth rates is found in normal cultured human mesothelial (NHM) cells derived from different donors. This variation is observed when the mesothelial cells are incubated in medium containing serum and when the potencies of several separate growth factors are measured by using defined media. Depending on the donor, gamma-interferon and interleukin-2 can be toxic, have no effect, or stimulate the growth rate of NHM cells. Cultured NHM cells can be induced to multiply by growth factors that are released by activated macrophages. Thus, interindividual variation in NHM cell growth control could play a role in the pathogenesis of mesothelioma for a person exposed to asbestos.