Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels

A polyamide nucleic acid (PNA) was designed by detaching the deoxyribose phosphate backbone of DNA in a computer model and replacing it with an achiral polyamide backbone. On the basis of this model, oligomers consisting of thymine-linked aminoethylglycyl units were prepared. These oligomers recognize their complementary target in double-stranded DNA by strand displacement. The displacement is made possible by the extraordinarily high stability of the PNA-DNA hybrids. The results show that the backbone of DNA can be replaced by a polyamide, with the resulting oligomer retaining base-specific hybridization.

https://science.sciencemag.org/content/sci/254/5037/1497.full.pdf

Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide

In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Directed evolution of novel binding proteins

The cloning of genes encoding mammalian DNA binding transcription factors for RNA polymerase II has provided the opportunity to analyze the structure and function of these proteins. This review summarizes recent studies that define structural domains for DNA binding and transcriptional activation functions in sequence-specific transcription factors. The mechanisms by which these factors may activate transcriptional initiation and by which they may be regulated to achieve differential gene expression are also discussed.

https://science.sciencemag.org/content/sci/245/4916/371.full.pdf

Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins

The zinc finger DNA-binding motif occurs in many proteins that regulate eukaryotic gene expression. The crystal structure of a complex containing the three zinc fingers from Zif268 (a mouse immediate early protein) and a consensus DNA-binding site has been determined at 2.1 angstroms resolution and refined to a crystallographic R factor of 18.2 percent. In this complex, the zinc fingers bind in the major groove of B-DNA and wrap part way around the double helix. Each finger has a similar relation to the DNA and makes its primary contacts in a three-base pair subsite. Residues from the amino-terminal portion of an alpha helix contact the bases, and most of the contracts are made with the guanine-rich strand of the DNA. This structure provides a framework for understanding how zinc fingers recognize DNA and suggests that this motif may provide a useful basis for the design of novel DNA-binding proteins.

https://science.sciencemag.org/content/sci/252/5007/809.full.pdf

Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.

Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy

A stretch of 200 kilobases (kb) of DNA from the I region of the mouse major histocompatibility complex has been cloned and characterized. It contains the genes for the biochemically defined class II proteins Eα, Eβ and Aβ. DNA blot analyses suggest that the I region may contain only 6–8 class II genes. Correlation of our molecular map with the genetic map of the I region confines two of the five I subregions, I–J and I–B, to less than 3.4 kb of DNA at the 3′ end of the Eβ gene where a hotspot for recombination has been observed. Indeed, the I–A and I–E subregions may be contiguous. If so, the I–B and I–J subregions are not encoded in the I region between the I–A and I–E subregions.

A molecular map of the immune response region from the major histocompatibility complex of the mouse

https://www.cell.com/cell/pdf/0092-8674(83)90536-6.pdf

Characterization of cloned cDNA representing rat myelin basic protein: Absence of expression in brain of shiverer mutant mice

Protein: absence of expression in brain of shiverer mutant mice.

In the proposed "zinc finger" DNA-binding motif, each repeat unit binds a zinc metal ion through invariant Cys and His residues and this drives the folding of each 30-residue unit into an independent nucleic acid-binding domain. To obtain structural information, we synthesized single and double zinc finger peptides from the yeast transcription activator ADR1, and assessed the metal-binding and DNA-binding properties of these peptides, as well as the solution structure of the metal-stabilized domains, with the use of a variety of spectroscopic techniques. A single zinc finger can exist as an independent structure sufficient for zinc-dependent DNA binding. An experimentally determined model of the single finger is proposed that is consistent with circular dichroism, one- and two-dimensional nuclear magnetic resonance, and visual spectroscopy of the single-finger peptide reconstituted in the presence of zinc.

Suzanna J. Horvath

Papers

Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy

A molecular map of the immune response region from the major histocompatibility complex of the mouse

Characterization of cloned cDNA representing rat myelin basic protein: Absence of expression in brain of shiverer mutant mice

Protein: absence of expression in brain of shiverer mutant mice.

Zinc-dependent structure of a single-finger domain of yeast ADR1.