A new method for sequencing DNA. 1977.
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Base Sequence↗
Biomedical subjects
A M Maxam
Publications and source records attributed to A M Maxam.
Identification of tissue specific nuclear proteins: DNA sequence and protein binding regions in the T cell receptor beta J-C intron.
We have determined the DNA sequences in the J2-C2 intron of the T cell receptor (TCR) beta gene and analyzed nuclear proteins binding to this region. Previously, we identified two tissue-specific DNase I hypersensitive regions, potential regulatory regions, in the J-C intron. The DNA sequence of the J2-C2 intron revealed that both DNase I hypersensitive regions have similar DNA sequences, suggesting that these regions are evolutionarily conserved. We have also identified tissue-specific nuclear-protein binding regions downstream of the DNase hypersensitive regions. Although transcriptional enhancer activity was not observed in the hypersensitive regions or the adjacent protein binding regions in the J-C intron, our findings suggest that the TCR-beta J-C intron may contain some other type of regulatory element.
Animals↗
The T cell receptor V alpha 3 gene segment is associated with reactivity to p-azobenzenearsonate.
The murine T cell receptor V alpha 3 gene segment is associated with reactivity to p-azobenzenearsonate, as indicated by several independent lines of evidence. First, three out of four arsonate-reactive T cell clones tested (two I-Ad-and one I-Ak-restricted) utilized V alpha 3. Second, bulk splenic cultures enriched for arsonate/H-2d and arsonate/H-2k responsive T cells showed increased expression of V alpha 3 mRNA. Third, a V alpha 3-containing alpha chain (Ar-5: arsonate/I-Ad) transferred arsonate responsiveness to an appropriate recipient T cell (O3: ovalbumin/I-Ad). Fourth, an independently derived V alpha 3-expressing T cell clone (2C: alloreactive to Ld) showed a response to arsonate/Ld. Thus, a V alpha 3 gene segment, in conjunction with at least two different J alpha segments (J alpha 20'(Ar-5) and J alpha pHDS58(2C)) and at least three different beta chains (V beta 2(Ar-5), V beta 6(O3), and V beta 8(2C], confers reactivity to arsonate in association with at least three different MHC proteins (I-Ad, I-Ak, and Ld). We suggest that V alpha 3 encodes a protein sequence with a binding site for the arsonate hapten.
Animals↗
T-cell antigen-receptor genes in autoimmune mice.
The developmental patterns of rearrangement and expression of the T-cell antigen-receptor genes are precisely regulated during T-cell differentiation and education. The beta- and gamma-subunit RNAs of the T-cell receptor are abundantly expressed in immature thymocytes. In mature thymocytes the alpha- and beta-subunit RNAs are preferentially expressed, whereas there is minimal expression of the gamma RNA. Although aspects of the pattern of known organization and rearrangement of the T-cell receptor gene in the thymus have been studied and the concept of a thymus selection process generally has been accepted, the cellular and molecular basis of thymus education remains obscure. Certain strains of mice with predilections for autoimmunity demonstrate T-cell developmental abnormalities. This is especially true for the lpr/lpr or gld/gld genotypes, in which the major population of peripheral T cells is developmentally disturbed. We have studied the development, expression, and rearrangement of T-cell receptor genes in the C3H/HeJ gld/gld mouse. Our results indicate a high level of expression of the beta and alpha RNAs in C3H/HeJ gld/gld T cells residing in the periphery. In addition, the beta-subunit gene of gld/gld peripheral T cells undergoes more rearrangements than does its normal C3H/HeJ T-cell counterparts. We speculate that this rearrangement pattern and high level of T-cell receptor mRNA reflects an abnormality or deficit in a thymus selection process that permits emigration of T cells with nonfunctionally rearranged T-cell receptor genes to secondary lymphoid organs. However, the normal level of gamma-subunit RNA expression argues that gamma-subunit gene rearrangements are distinct from processes related to alpha- and beta-subunit selection.
Animals↗
Active T-cell receptor genes have intron deoxyribonuclease hypersensitive sites.
The T-cell receptor beta-chain gene has a nuclease hypersensitive site in several kinds of T cells, which does not appear in B cells expressing immunoglobulins. Conversely, the kappa immunoglobulin gene shows a known hypersensitive site at its enhancer element in B cells, as expected, but this site is absent in T cells. As is the case with immunoglobulin genes, the T-cell receptor site lies within the gene, in the intron separating joining and constant region segments. These nuclease hypersensitive DNA configurations in the introns of active T-cell receptor and immunoglobulin genes may arise from control elements that share ancestry but have diverged to the extent that each normally acts only in lymphoid cells which use the proximal gene product.
Animals↗
Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis.
The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.
Base Sequence↗
Sequencing end-labeled DNA with base-specific chemical cleavages.
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Base Sequence↗
Sequencing the DNA of recombinant chromosomes.
With contemporary molecular cloning and DNA sequencing techniques, deriving the primary structure of higher cell genes is now routine. This publication reviews the chemical DNA sequencing method, and suggests strategies for sequencing recombinant DNA, whether arising naturally by chromosome rearrangement in vivo or created experimentally by gene splicing in vitro. One such strategy prepares end-labeled restriction fragments from an inserted or rearranged DNA region, for sequencing by the chemical method. Another maps the point at which the sequences of two related DNA regions diverge, and indicates which restriction endonucleases would be useful for sequencing across that point. These techniques can facilitate sequencing of DNA integrations, excisions, translocations, inversions, and introns in cloned chromosomal segments.
Animals↗
Structure mapping of 5'-32P-labeled RNA with S1 nuclease.
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Base Sequence↗
Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence.
The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.
Base Sequence↗
Sequence of a mouse germ-line gene for a variable region of an immunoglobulin light chain.
We have determined the sequence of the DNA of a germ-line gene for the variable region of a mouse immunoglobulin light chain, the VlambdaII gene. The sequence confirms that the variable region gene lies on the DNA separated from the constant region. Hypervariable region codons appear in the germ-line sequence. A sequence for the hydrophobic leader, 19 amino acids that are cleaved from the amino terminus of the protein, appears near, but not continuous with, the light chain structural sequence: most of the leader sequence is separated from the rest of the gene by 93 bases of untranslated DNA.
Animals↗
Detection of 5-methylcytosine in DNA sequences.
Col E1 DNA has methylated cytosine in the sequence 5'-CC*(A/T)GG-3' and methylated adenine in the sequence 5'-GA*TC-3' at the positions indicated by asterisks(*). When the Maxam-Gilbert DNA sequencing method is applied to this DNA, the methylated cytosine (5-methylcytosine) is found to be less reactive to hydrazine than are cytosine and thymine, so that a band corresponding to that base does not appear in the pyrimidine cleavage patterns. The existence of the methylated cytosine can be confirmed by analyzing the complementary strand or unmethylated DNA. In contrast, the methylated adenine (probably N6-methyladenine) cannot be distinguished from adenine with standard conditions for cleavage at adenine.
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Location of the 5.8S rRNA gene of Saccharomyces cerevisiae.
Direct DNA sequence analysis of Saccharomyces cerevisiae ribosomal DNA cloned in an Escherichia coli plasmid revealed part of the structural gene for 5.8S rRNA at one end of a 700-base-pair EcoRI fragment. Taken with the previously established EcoRI restriction map of the ribosomal repeat unit, this sequence establishes that the yeast 5.8S RNA segment is located between the 18S and 28S segments in the 42S rRNA precursor and in the DNA which codes for it.
Base Sequence↗
Promotor region for yeast 5S ribosomal RNA.
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Base Sequence↗
A new method for sequencing DNA.
DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.
Adenine↗
Rous sarcoma virus genome is terminally redundant: the 5' sequence.
When Rous sarcoma virus RNA is transcribed into DNA by the reverse transcriptase, a tRNA primer is elongated into DNA. The primer is near the 5' end of the virus genome; the first major DNA made is a "run-off" product extending 101 bases from the primer to the 5' end of the template. We have studied this DNA molecule to determine the sequence of the first 101 bases at the 5' end of the Rous sarcoma virus genome (Prague strain, subgroup C). Twenty-one bases at the extreme 5' end are also at the 3' end of the virus genome (see D. E. Schwartz, P. C. Zamecnik, and H. L. Weith, this issue, pp. 994-998), and thus this virus is terminally redundant. The existence of this sequence repetition immediately suggests mechanisms by which the growing DNA copy can jump from the 5' end to a 3' end of the template and become circular. The sequence also displays a possible ribosome binding site and enough secondary structure to permit a possible 5'-5' linkage of viral RNA molecules.
Avian Sarcoma Viruses↗
Mapping adenines, guanines, and pyrimidines in RNA.
The positions of adenines, guanines, and pyrimidines can be determined by partial nuclease digestion of a terminally labeles RNA molecule. In urea, at elevated temperatures, RNase T1 generates a pattern reflecting cleavage at guanines while RNase U2 cleaves only at adenine. A limited alkaline hydrolysis provides a continuum of fragments derived from breaks at every phosphodiester bond. The reaction products are electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel. An autoradiograph of the gel displays the sequence up to 100 nucleotides from the end of the molecule, although uracil cannot as yet be distinguished from cytosine. These techniques form the basis of an RNA sequencing method and are demonstrated on yeast 5.8S ribosomal RNA.
Adenine↗
Enzymatic in vitro synthesis of globin genes.
Full-length, single-stranded rabbit globin cDNA, synthesized by AMV reverse transcriptase, apparently contains a small double-stranded sequence (hairpin) at the 3' terminus. This cDNA can serve as template-primer for E. coli DNA polymerase I, which synthesizes a strand complementary to the cDNA and covalently bound to it. The loop connecting the two strands can be cut by S1 nuclease. Reassociation, hybridization, and restriction endonuclease studies, as well as electrophoretic analyses, indicate that the sequential actions of reverse transcriptase, DNA polymerase 1, and S1 nuclease generate full-length, double-stranded synthetic globin genes.
Animals↗