We describe a rapid "nonrandom" DNA sequence analysis procedure that facilitates the nucleotide sequence determination of large contiguous regions of DNA. The method consists of cloning a restriction endonuclease fragment of interest into bacteriophage M13 followed by construction of a series of nuclease BAL-31 deletion mutants originating from a single site in M13 that is close to the DNA insert. Determination of the size of the deletion mutant is accomplished by hybridization to a complementary single-stranded probe derived from M13 containing the total insert followed by nuclease SI treatment. Single-stranded M13-insert DNAs of progressively smaller sizes are isolated and analyzed by using a site-specific M13 DNA primer and the dideoxy chain-termination method. In this way, analysis of the DNA sequence proceeds from one -end of the total insert to the other in a nonrandom fashion due to generation ofa controlled overlapping set of deletion mutants.Advances in DNA sequence analysis techniques have revolutionized the study of cloned gene structure (1-3). The most commonly used method for DNA sequence analysis has been the chemical degradation procedure (1). There are several disadvantages to this technique because prior knowledge of the restriction endonuclease map of the fragment is required for detailed formulation of an analysis strategy. In addition, end labeling and sequence analysis from a single 5' or 3' end necessitates the use of relatively large amounts of purified fragment, a radioisotope of high specific activity, and relatively long exposure times for sequence readings.The use of bacteriophage M13 for subcloning and DNA sequence analysis offers distinct advantages in overcoming some of these difficulties. A number of recent reports describe the use of M13 for"random" DNA sequence analysis by the dideoxy chain-termination method using an M13 site-specific primer (4,5). This bacteriophage is well suited for DNA sequence analysis by the dideoxy chain-termination method, because cloning and isolation ofrecombinants is rapid and single-stranded phage DNA of he "+" strand covalently linked to single-stranded insert DNA is easily isolated from the culture medium. DNA sequence analysis is then accomplished by-the dideoxy chain-termination -method using the single-stranded M13 (+) insert DNA and a site-specific M13 primer that hybridizes close to the insert. One limitation of this technique is that only several hundred nucleotides from the primer site can be reliably read from gels. Therefore random analysis of a large DNA fragment involves the use of several restriction enzymes to develop a series ofsmall overlapping fragments that are subcloned into bacteriophage M13 and then analyzed. The entire DNA sequence is then assembled by matching of overlapping sequences, frequently with the aid of a computer. Another disadvantage of this random method is unnecessary redundancy in analysis; some regions may be analyzed several times before the entire DNA sequence can be assembled. Potential difficulties ...

Polypeptides which are immunologically related to the erythrocyte anion transport protein have been identified in a variety of non‐erythroid cells. We describe two cDNA clones encoding a human non‐erythroid band 3 protein (HKB3) and the mouse erythrocyte band 3 (MEB3) and show that these proteins are structurally similar. Comparison of the predicted amino acid sequences from HKB3 and MEB3 reveals a high degree of sequence homology (71%) and conservation of the overall topography of the transmembrane domain. Similar levels of homology are also observed in comparisons with published amino acid sequence from the human erythrocyte band 3. In addition, specific residues which have been demonstrated to be involved in erythroid anion transport are conserved in HKB3, suggesting that this non‐erythroid band 3 protein functions in this respect. Although protein sequence homology within the cytoplasmic domain is considerably lower (35%), three specific regions in HKB3 are conserved, one of which may represent an ankyrin binding site. Northern blot analysis reveals transcripts that cross‐hybridize with the HKB3 cDNA in a variety of non‐erythroid cell lines but not in cells of erythroid lineage.

We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cell line that carries a reciprocal translocation between chromosomes 8 and 14. The breakpoint lies within the first intron of c-myc, so that the first noncoding exon of the gene remains on the 8q- chromosome. The second and third coding exons are translocated to the 14q+ chromosome into the switch region of C-alpha 1. The orientation of the c-myc gene with relationship to alpha 1 is 5' to 5', with directions of transcription in opposite orientation. DNA sequencing studies predict five changes in the amino acid sequence of the myc protein, two of which occur in a region within the second exon which is highly conserved in evolution. Southern blotting data indicate that the first exon of c-myc is rearranged 3' to 3' with the pseudo-epsilon gene. Because CA46 cells contain two rearranged mu genes, the translocation must have occurred after immunoglobulin rearrangement. The position of the breakpoint in CA46 occurs within a 20-base-pair region of the first intron of c-myc to which breakpoints have been mapped for two additional B-cell lymphomas with the t(8;14) translocation, ST486 and the Manca cell line. The region of the heavy chain locus to which c-myc has translocated is different in each case. Comparisons have been made of the levels of transcripts of the translocated c-myc gene in ST486 and CA46, where the gene is not associated with the heavy chain enhancer, with its expression in the Manca cell, in which it is. The c-myc gene is transcribed at similar levels in all three cases.

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