Donna L. Daniels

Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76.0 to 81.5 minutes

The DNA sequence of a 225.4 kilobase segment of the Escherichia coli K-12 genome is described here, from 76.0 to 81.5 minutes on the genetic map. This brings the total of contiguous sequence from the E.coli genome project to 725.1 kb (76.0 to 92.8 minutes). We found 191 putative coding genes (ORFs) of which 72 genes were previously known, and 110 of which remain unidentified despite literature and similarity searches. Seven new genes--arsE, arsF, arsG, treF, xylR, xylG, and xylH--were identified as well as the previously mapped pit and dctA genes. The arrangement of proposed genes relative to possible promoters and terminators suggests 90 potential transcription units. Other features include 19 REP elements, 95 computer-predicted bends, 50 Chi sites, and one grey hole. Thirty-one putative signal peptides were found, including those of thirteen known membrane or periplasmic proteins. One tRNA gene (proK) and two insertion sequences (IS5 and IS150) are located in this segment. The genes in this region are organized with equal numbers oriented with or against replication.

Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli

Sequences of four new heat-shock (HS) genes of Escherichia coli organized into two operons were determined. The operon at 83 min specifies two proteins of 15.8 kDa (HslT) and 16.1 kDa (HslS), which are identical to IbpA and IbpB, respectively. Expression of mRNA from a sigma 32-dependent promoter of the hslTS/ibpAB operon is stimulated 30-75-fold upon temperature upshift. The transcription start point (tsp) is located at a G, 96 bp upstream from the AUG start codon of hslT/ibpA. The deduced amino acid sequences of HslT/IbpA and HslS/IbpB are 48% identical to each other and were found to be remotely related to the chloroplast low-molecular-weight HS protein, which is highly conserved among plants. The second hs operon is much less actively stimulated by temperature upshift, although it has a hs promoter that perfectly matches the consensus of promoters recognized by sigma 32. Located at 88.9 min, the hslVU operon specifies proteins of 19.1 kDa (HslV) and 49.6 kDa (HslU). Multiple tsp were found in this operon. HslV is remotely related to the eukaryotic proteasome proteins, and HslU is very similar to a Pasteurella haemolytica protein of unknown function. Both HslU and the P. haemolytica protein share a ATP/GTP-binding motif near their N-termini. The two operons described here are transcribed counterclockwise on the standard genetic map.

Nucleotide sequence of the Q gene and the Q to S intergenic region of bacteriophage lambda

Abstract The Q gene of bacteriophage lambda codes for a positive regulator which is responsible for turning on late transcription during lytic growth. We have determined the nucleotide sequence of this gene as well as the region between genes Q and S , which includes a site of Q action. The Q protein is predicted by its nucleotide sequence to have a molecular weight of 22.5 (207 amino acids). It appears to have no homology with either N or the sigma subunit of RNA polymerase. The Q to S intergenic region includes the 6 S RNA coding region, the late promoter (p′ r ) and an open reading frame indicating a possible new gene of unknown function which we term orf-64. The orf-64 protein product would have a molecular weight of 7083 (64 amino acids). It starts within the 6 S RNA coding sequence and extends 11 amino acids beyond it and suggests a possible “leader peptide” function. No other peptide larger than 15 residues could be coded by the DNA between Q and S . The position of the S gene was located by mapping a Tn5 insertion in gene S with restriction endonucleases. The distance between Q and S is 672 nucleotides and it is not known where within this large area Q acts. Analysis of the sequence for inverted repeats revealed a number of possible loop-stem structures which could be involved in transcription termination, RNA processing, or Q-mediated transcription activation.

Q-mediated late gene transcription of bacteriophage λ: RNA start point and RNase III processing sitesin Vivo

The location of the RNA start point of in vivo Q-activated late gene RNA of bacteriophage lambda has been determined to be identical to the start point of in vitro 6 S RNA. The 6 S RNA is made early in infection and is efficiently antiterminated by Q. Two RNase III cut sites are located within Q-dependent RNA sequences, 209 and 270 bp from the beginning of the late transcript, and lie on the stem of an inverted repeat which has features in common with previously described RNase III processing sites. This is the third example of RNase III cut sites immediately downstream of transcription termination points in lambda, the others being antiterminated N gene mRNA and int gene mRNA.

Simultaneous preparation of up to 768 single-stranded DNAs for use as templates in DNA sequencing

A method for simultaneously preparing large numbers of single-stranded M13 DNAs for use as templates in enzymatic chain-termination sequencing is described. We have optimized phage growth in microtiter dish wells and adapted the DNA extraction method of I. C. Eperon (1986, Anal. Biochem. 156 , 406–412) and D. Cohen (personal communication). Its use in a high-throughput genomic sequencing facility is discussed. The procedure yields a 30-fold increase in throughput relative to classic methods for this critical and labor-intensive step of the sequencing process. The method is inexpensive and requires only commercially available equipment and supplies, making it useful in individual laboratories as well as for a large project.

APPENDIX I A Molecular Map of Coliphage Lambda

The molecular map of λ is presented in the following sections: Figure 1 is a scale drawing of the λ map. Table 1 presents our tabulation of all the sites on the λ map. Listed are the names of all mapped sites (columns A and E), their base-pair coordinates on the 5′–3′ l strand of λ DNA (column C), four-character symbols used by the computer for sequence annotation in Appendix II (column D), a description of the site (columns F and H), and the method of localization on the map (column G). Restriction sites that have been mapped experimentally are shown in column A. All other sites are specified in column E. Table 2 lists the published sources of λ DNA sequence. Table 3 shows the sources of mapping information for sites other than genes. Table 4 shows the sources of mapping information for protein-coding genes and open reading frames (orfs). Appendix II presents the complete sequence of λ with all mapping information. SOURCES The sources of information used to compile the molecular map of phage λ DNA areThe complete nucleotide sequence of λ . Various groups have sequenced regions of different strains of λ . This information has been collated, and sequences have been compared from all sources to determine overlaps and discrepancies. This information forms eight “sequence blocks,” which are indicated in Table 1 (column B) by ditto marks enclosed by the symbols ▿ and ▵. Within these blocks are only four unresolved discrepancies (single base-pair changes).