Specific Recognition Capability Research Articles

Bioengineering and characterization of DNA-protein assemblies floating on supported membranes.

A biodevice involving thiolated ssDNA and engineered cytochrome b5 linked through a cis-platine bridge is described. This original nanostructure is associated to a supported membrane through a floating anchor, thus constituting a dynamic bidimensionnal DNA capture device contrasting with the constrained geometry of currently available DNA chips. Characterization by optical spectroscopy, surface plasmon resonance and gel electrophoresis demonstrated that an unique molecular supra-assembly featuring specific DNA recognition capability has been obtained. This device is characterized by the reversibility of its assembly, self-organization and fluidity properties and is of interest as a prototype to design new generations of DNA chip biosensors.

Zinc fingers: Gilt by association

A major focus of molecular biology is the identification of mechanisms by which eukaryotic gene transcription is regulated in a precise temporal and spatial fashion. The identification of transcriptional factors with the ability to recognize and bind to specific DNA sequences has provided important clues to elucidating these mechanisms. X-ray crystallographic analysis of prokaryotic DNA binding proteins such as lambda repressor and cro revealed a common helix-turn-helix motif responsible for their interaction with DNA(Pabo and Sauer, Ann. Rev. Biothem. 53, 293-321, 1984). The notion that this structure is universally used for specific DNA interaction was challenged by Klug’s recognition of a new DNA binding motif in Xenopus laevis transcription factor TFIIIA: the zinc finger (Miller et al., EMBO J. 4, 1609-1614, 1985). Although originally recognized in an RNA polymerase Ill transcription factor, recent analysis has revealed potential metal binding fingers as a surprisingly common structure in a variety of nucleic acid binding proteins (for review see Berg, Science 232,485-487, 1986), including factors likely to influence polymerase II transcription. Finger Structure and Metal Binding Isolation and comparison of cDNAs encoding potential finger proteins indicate the existence of at least two classes of proteins with specific nucleic acid recognition capabilities. These classes are characterized according to the number and position of the cysteine and histidine residues available for zinc coordination (see table). The &HZ class is typified by TFIIIA and is comprised of proteins containing, as the basic structural unit, pairs of cysteines and histidines separated by a loop of twelve amino acids. Although most easily visualized as a finger of amino acids anchored by zinc and projecting from the surface of the protein (see figure, part A), an alternate view, inferred from structures of known metalloproteins, suggests the more complex model depicted in part B of the figure (Berg, PNAS 85, 99-102, 1988). In the C2H2 proteins identified, the finger motif is tandemly repeated a minimum of two times with a 7 to 8 amino acid linker separating the units (the H/C link). In contrast to the C2H2 group, the C, proteins have a variable number of conserved cysteines available for metal chelation. For the yeast transcriptional factors, such as GAL4, the putative finger region exists as a cluster of six invariant cysteines (C, family), whereas the steroid receptors contain two apparently unrelated fingers encoded by separate exons (Huckaby et al., PNAS 84, 8380-8384, 1987) with four (C,) and five (C,) conserved Minireview

Sequence of rat liver alpha 2-macroglobulin and acute phase control of its messenger RNA.

Six alpha 2-macroglobulin cDNA clones were isolated from two liver cDNA libraries produced from rats undergoing acute inflammation. The coding sequence for rat alpha 2-macroglobulin including its 27-residue signal peptide and the 3' - and part of the 5' nontranslated regions were determined. The mature protein consisting of 1445 amino acids is coded for by a 4790 +/- 40 nucleotide messenger RNA. It contains a typical internal thiol ester region and 25 cysteine residues which are conserved between rat and human alpha 2-macroglobulin. Although the amino acid sequences of rat and human alpha 2-macroglobulin share 73% identity, two small divergent areas of 17 and 38 residues were found, corresponding to 29 and 11% identity, respectively. These areas are located in the bait region and, therefore, may confer specific proteinase recognition capabilities on rat alpha 2-macroglobulin. Following an inflammatory stimulation, rat alpha 2-macroglobulin mRNA levels increased 214-fold over control values and reached a maximum at 18 h. By 24 h the levels had decreased to less than 30% of the maximum value. Transcription rates from the alpha 2-macroglobulin gene as measured in nuclear run-on experiments showed a less than 3-fold increase in nuclei from acutely inflamed rats as compared to controls. These results suggest that the accummulation of alpha 2M mRNA is due to the combined effects of increased transcription rates and post-transcriptional processing.