Walter Gilbert

[57] Sequencing end-labeled DNA with base-specific chemical cleavages

Publisher Summary This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.

Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis

We have discovered that single-stranded DNA containing short guanine-rich motifs will self-associate at physiological salt concentrations to make four-stranded structures in which the strands run in parallel fashion. We believe these complexes are held together by guanines bonded to each other by Hoogsteen pairing. Such guanine-rich sequences occur in immunoglobulin switch regions1, in gene promoters2,3, and in chromosomal telomeres4. We speculate that this self-recognition of guanine-rich motifs of DNA serves to bring together, and to zipper up in register, the four homologous chromatids during meiosis.

E. coli RNA polymerase interacts homologously with two different promoters.

We present and review experiments that identify points of close approach of the RNA polymerase to two promoters, lac UV5 and T7 A3. We identify the contacts to the phosphates along the DNA backbone, to the N7s of guanines in the major groove and the N3s of adenines in the minor groove, and to the methyl groups of thymines. These contacts to the two promoters are strikingly homologous in space, as shown on three-dimensional models, and identify major regions of interactions lying on one side of the DNA molecule (at -35 and -16), as well as further areas extending through the Pribnow box. Both promoters are unwound similarly by the polymerase, across a region of about twelve bases extending from the middle of the Pribnow box to just beyond the RNA start site. We discuss the areas of interaction in the context of promoter homologies and promoter mutations. The disposition of the contacts in space suggests a model for the pathway along which the RNA polymerase binds to promoters.

The structure and evolution of the two nonallelic rat preproinsulin genes

In the rat, there are two nonallelic genes for preproinsulin. The insulin end products are very similar and are equally expressed. We have isolated clones carrying these genes and their flanking sequences, and characterized them by DNA sequencing and electron microscopic analysis. We have established the primary structure of the preproinsulin mRNAs and the signal peptides of these two proteins. One of the genes contains two introns: a 499 bp intron interrupting the region encoding the connecting peptide and a 119 bp intron interrupting the segment encoding the 5 noncoding region of the mRNA. The introns are transcribed and present in a preproinsulin mRNA precursor. The other gene possesses the smaller, but not the larger, of the two introns. Calculations based on the divergence of the two preproinsulin nucleotide and amino acid sequences indicate that these genes are the products of a recent duplication. Thus one of the genes gained or lost an intron since that time.

Polypeptide synthesis in Escherichia coli: II. The polypeptide chain and S-RNA

The polyphenylalanine chain, made by poly U-directed† synthesis in the cell-free system from Escherichia coli , is studied. This nascent polypeptide chain is bound to the 50 s subunit of the 70 s ribosome. Furthermore, the growing chain is covalently linked to an S-RNA molecule. The bond between the polypetide chain and the S-RNA is similar to that in amino acyl S-RNA but is an order of magnitude more stable against alkaline hydrolysis or hydroxylamine treatment. When puromycin releases the chain from the ribosome, it breaks the bond to the S-RNA. This terminal S-RNA is used as an end group method to measure the molecular weight of the nascent chain. Chains of the order of 40 amino acids are made, and new chains are initiated throughout the reaction. The behavior of the nascent proteins found after cell-free synthesis in E. coli differs from that of the polyphenylalanine only in that the bound nascent proteins stabilize the 70 s ribosome to a higher degree and bind less tightly to the 50 s subunit at low magnesium ion concentrations.

Large-scale comparison of intron positions among animal, plant, and fungal genes

We purge large databases of animal, plant, and fungal intron-containing genes to a 20% similarity level and then identify the most similar animal–plant, animal–fungal, and plant–fungal protein pairs. We identify the introns in each BLAST 2.0 alignment and score matched intron positions and slid (near-matched, within six nucleotides) intron positions automatically. Overall we find that 10% of the animal introns match plant positions, and a further 7% are “slides.” Fifteen percent of fungal introns match animal positions, and 13% match plant positions. Furthermore, the number of alignments with high numbers of matches deviates greatly from the Poisson expectation. The 30 animal–plant alignments with the highest matches (for which 44% of animal introns match plant positions) when aligned with fungal genes are also highly enriched for triple matches: 39% of the fungal introns match both animal and plant positions. This is strong evidence for ancestral introns predating the animal–plant–fungal divergence, and in complete opposition to any expectations based on random insertion. In examining the slid introns, we show that at least half are caused by imperfections in the alignments, and are most likely to be actual matches at common positions. Thus, our final estimates are that ≈14% of animal introns match plant positions, and that ≈17–18% of fungal introns match animal or plant positions, all of these being likely to be ancestral in the eukaryotes.

Polypeptide synthesis in Escherichia coli. I. Ribosomes and the active complex.

The poly U-directed† synthesis of polyphenylalanine in the cell-free system from Escherichia coli is used as a model system in which to investigate the interaction of messenger RNA with ribosomes. It is shown that both the 50 s and the 30 s ribosome are necessary for polypeptide synthesis. The ribosomes accept the messenger RNA under conditions in which the dominant form is the 70 s particles, but the particles involved will dimerize more readily than the rest of the 70 s particles. All of the synthetic activity of the poly U-ribosome mixture appears as a rapidly sedimenting complex, 140 to 200 s. This active complex depends upon RNA for its integrity and contains an amount of poly U consistent with one molecule for several ribosomes. Furthermore, all of the synthetic capacity of the usual crude extract from E. coli is in the form of rapidly sedimenting complexes in the 100 to 200 s range.

THE BINDING OF S-RNA BY ESCHERICHIA COLI RIBOSOMES.

Washed ribosomes from E. coli will bind soluble RNA (S-RNA) in high concentrations of magnesium ions. The bound S-RNA will not wash off in high magnesium but can be displaced by additional S-RNA or can be freed by lowering the magnesium ion concentration. This binding takes place in the cold and does not require the supernatant enzymes or an energy source. It is not affected either by two inhibitors of protein synthesis, puromycin and chloramphenicol, or by the charging of the S-RNA with amino acids. The binding requires the integrity of the pCpCpA§ end of the S-RNA molecule. There is one site for this binding on the 70 s ribosome, on the 50 s subunit. After protein synthesis in a cell-free extract from E. coli , a small fraction of the S-RNA that is bound to the ribosomes in high magnesium becomes resistant to being washed off the ribosomes in low magnesium. This tightly bound fraction appears on the 50 s subunit of the “stuck” 70 s ribosome (those 70 s ribosomes that do not dissociate in low magnesium and that carry nascent polypeptide chains). It is resistant to ribonuclease under mild conditions. This bound S-RNA can be recovered as intact 4 s material if the ribosomes are broken up with sodium dodecyl sulfate.

The triosephosphate isomerase gene from maize introns antedate the plant-animal divergence

We have cloned and characterized a cDNA and genomic DNA for the triosephosphate isomerase expressed in maize roots. The gene is interrupted by eight introns. If we compare this gene with that for the protein in chicken, which has six introns, we see that five of the introns are at identical places, one has shifted by three codons, and two are totally new. This great matching leads us to conclude that the introns were in place before the plant-animal divergence, and that the parental gene had at least eight introns, two of which were lost in the line that leads to animals.

On the ancient nature of introns

We discuss some of the arguments for introns arising early or late in evolution. We outline the exon theory of genes and discuss the series of discoveries of introns in the gene (TPI) encoding triosephosphate isomerase (TPI) that have filled out a series of better fits to the Go plot, culminating in the 1986 prediction of an intron position that was finally discovered in 1992. We present a statistical argument that the 11-intron structure of TPI (based on attributing all of the introns to an ancestral gene and interpreting three cases of very close intron positions as examples of sliding) has a clear relationship to the protein structure. The exons of this 11-intron TPI are a better approximation to Mitiko Gos modules (Go, 1981) than are 99.9% of all alternative exon patterns corresponding to 11 introns placed randomly in the gene, and better than 96% of all alternative patterns in which the lengths of the exons are preserved while the introns are moved. We combine four tests relating exons to protein structure: (i) whether the exons are compact modules, (ii) whether the exons contain most of the close contacts in the protein, (iii) whether the exon configuration maximized buried surface area along the backbone, and (iv) whether the exons maximize their content of hydrogen bonds. On a joint measure for these tests, the native exon structure with 11 introns fits these tests better than 99.4% of all alternative structures obtained by permuting the exon lengths and intron positions.(ABSTRACT TRUNCATED AT 250 WORDS)