Biomedical subjects
G Sarkar
Publications and source records attributed to G Sarkar.
Shedding light on PCR contamination.
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Recurrent nonsense mutations at arginine residues cause severe hemophilia B in unrelated hemophiliacs.
Direct sequencing of the regions of the factor IX gene of likely functional significance was performed in four patients with severe hemophilia B. In two of the individuals, a transition at the dinucleotide CpG caused a nonsense mutation at arginine 333. In the other two individuals, a transition at CpG caused a nonsense mutation at arginine 29. Since these patients are all unrelated, as shown by differing alleles of the TaqI polymorphism in intron four or extensive nonoverlapping pedigrees, the mutations arose independently. In addition, the origin of one arginine 333 mutation in one family has been traced to the germline of the maternal grandfather. The frequent occurrence of mutations at arginine codons that contain the sequence CGN can be explained by the dramatic elevation of transitions at CpG. As a result, approximately one in four individuals with hemophilia B is expected to have a mutation at arginine and nonsense mutations at one of six arginine residues should be common causes of severe hemophilia.
Characterization of polymerase chain reaction amplification of specific alleles.
Under certain conditions, polymerase chain reaction (PCR) can be used to differentially amplify one allele over another. To characterize the phenomenon, we have made a series of PCR primers and determined whether differential amplification could be detected after agarose gel electrophoresis. Two allele pairs were examined; one pair represents a transversion and one pair represents a transition. The following conclusions emerge: (i) when the 3' or the 3' penultimate base of the oligonucleotide mismatched an allele, no amplification product could be detected; (ii) when the mismatches were 3 and 4 bases from the 3' end of the primer, differential amplification was still observed, but only at certain concentrations of magnesium chloride; (iii) the mismatched allele can be detected in the presence of a 40-fold excess of the matched allele; (iv) primers as short as 13 nucleotides were effective; and (v) the specificity of the amplification could be overwhelmed by greatly increasing the concentration of target DNA.
Direct sequencing of the activation peptide and the catalytic domain of the factor IX gene in six species.
By means of RNA amplification with transcript sequencing (RAWTS) under low stringency conditions, sequence was obtained directly without cloning for the activation peptide and the catalytic domain of factor IX from six species--sheep, pig, rabbit, guinea pig, rat, and mouse. The data presented demonstrate that, by the appropriate design of oligonucleotides and by performance of a nested PCR under appropriate conditions, it is possible to obtain sequence on a battery of species with a minimum of oligonucleotide primers. A total of 5.2 kb of cross-species sequence was generated with RAWTS. The results indicate that (1) 69% of the amino acids in the catalytic domain, but only 23% of the amino acids in the activation peptide, are identical in humans and the six species; (2) the catalytic domain evolves at a slower rate, but the extent and pattern of conservation of amino acids in the activation peptide suggest that the peptide functions as more than a cleavage spacer that separates the heavy and light chains in the catalytically inactive zymogen; (3) 37% of the amino acids in the activation peptide and 34% of the amino acids in the catalytic domain are factor IX-specific; i.e., they are either identical or changed in a highly conservative fashion in factor IX, but not in other related coagulation proteases; (4) these conserved factor IX-specific amino acids fall into three clusters, which are candidates for involvement in the protein interactions specific to factor IX; (5) there is a human-specific deletion after lysine 142 and a rodent-specific insertion after alanine 161; (6) in guinea pig, the insertion is associated with a seven-amino-acid repeat that corresponds to a perfect repeat of a 21-bp sequence; (7) humans have lost a potential N-glycosylation site that is conserved in the other species; (8) in each species, a few nonconservative changes occur in amino acids that are otherwise completely conserved, suggesting that compensatory mutations may have occurred; and (9) when compared to that of mouse, the amino acid identity with guinea pig factor IX is no greater than that found for the non-rodent species, a result compatible with the postulated increased rate of evolution in rodents.
The "megaprimer" method of site-directed mutagenesis.
We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.
Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity.
RNA amplification with transcript sequencing (RAWTS) is a rapid and sensitive method of direct sequencing that involves complementary DNA synthesis, polymerase chain reaction (PCR) with a primer or primers containing a phage promoter, transcription from the phage promoter, and reverse transcriptase-mediated sequencing. By means of RAWTS, it was possible to sequence each of four tissue-specific human messenger RNAs (blue pigment, factor IX, phenylalanine hydroxylase, and tyrosine hydroxylase) in four cell types examined (white blood cells, liver, K562 erythroleukemia cells, and chorionic villus cells). These results indicate that there is a basal rate of transcription, splicing, and polyadenylation of tissue-specific mRNAs in adult and embryonic tissues. In addition to revealing sequence information, it is possible to generate a desired in vitro translation product by incorporating a translation initiation signal into the appropriate PCR primer. RAWTS can be used to obtain novel mRNA sequence information from other species as illustrated with a segment of the catalytic domain of factor IX. In general, the ability to obtain mRNA sequences rapidly across species boundaries should aid both the study of protein evolution and the identification of sequences crucial for protein structure and function.
Accurate prenatal diagnosis with novel polymerase chain reaction primers in a family with sporadic hemophilia A.
Novel oligonucleotide primers are described for the convenient and internally controlled detection of the BclI factor VIII polymorphism on agarose gels. The primers were used for prenatal diagnosis in a family in which only one individual was affected with hemophilia A. In such families, it is important for the mother to understand that uncertainty about the point of origin of the mutation precludes the accurate diagnosis of affected fetuses but does allow the accurate diagnosis of unaffected fetuses in many cases.
Specific amplification with PCR of a refractory segment of genomic DNA.
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RNA amplification with transcript sequencing (RAWTS).
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Genomic amplification with transcript sequencing.
A sequencing method called genomic amplification with transcript sequencing (GAWTS) is described that is based on amplification with the polymerase chain reaction (PCR). GAWTS bypasses cloning and increases the rate of sequence acquisition by at least fivefold. The method involves the attachment of a phage promoter onto at least one of the PCR primers. The segments amplified by PCR are transcribed to further increase the signal and to provide an abundance of single-stranded template for reverse transcriptase-mediated dideoxy sequencing. An end-labeled reverse transcriptase primer complementary to the desired sequence generates the additional specificity required to generate unambiguous sequence data. GAWTS can be performed on as little as a nanogram of genomic DNA. The rate of GAWTS can be increased by coamplification and cotranscription of multiple regions as illustrated by two regions of the factor IX gene. Since GAWTS lends itself well to automation, further increases in the rate of sequence acquisition can be expected.
Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP.
It has been suggested that the cap-binding protein complex is involved in ATP-mediated melting of 5'-mRNA secondary structure to facilitate ribosome binding during initiation of translation in eukaryotic cells (Edery, I., Lee, K. A. W., and Sonenberg, N. (1984) Biochemistry 23, 2456-2462). Consequently, we have studied the interaction of dATP/ATP with the eukaryotic cap-binding protein complex by UV photoaffinity labeling. UV irradiation of the cap-binding protein complex in the presence of [alpha-32P]dATP/ATP resulted in the cross-linking of this compound to the 50-kDa polypeptide of the complex. This polypeptide is almost identical to the previously characterized eukaryotic initiation factor (eIF) 4A. We examined the ability of dATP/ATP to cross-link to eIF-4A and found that it cross-links less efficiently (approximately 60-fold on a molar basis) compared to the cross-linking obtained for the eIF-4A component of the cap-binding protein complex. Irradiation of purified eIF-4A together with the cap-binding protein complex in the presence of [alpha-32P]dATP resulted in greater than additive labeling of the eIF-4A component of the cap-binding protein complex and purified eIF-4A, suggesting a synergistic interaction between purified eIF-4A, the cap-binding protein complex, and dATP/ATP. We also report that photoaffinity labeling of eIF-4A and the eIF-4A component in the cap-binding protein complex is stimulated by eIF-4B, but not by other initiation factors or mRNA.
Identification of a new polypeptide coded by reovirus gene S1.
The reovirus S1 gene has recently been shown potentially to encode two polypeptides (from two overlapping reading frames) having predicted molecular weights of 49,071 and 16,143 (Nagata et al., Nucleic Acids Res. 12:8699-8710, 1984; Bassel-Duby et al., Nature [London], in press). The larger polypeptide is reovirus protein sigma 1, but synthesis of the smaller polypeptide has not been described to date. A truncated clone of the S1 gene in which the first ATG is deleted was expressed in an in vitro protein synthesis system to yield a approximately 13-kilodalton polypeptide, as determined from migration on sodium dodecyl sulfate-polyacrylamide gels. A polypeptide with a similar migration pattern on sodium dodecyl sulfate-polyacrylamide gels was present in reovirus-infected cells and absent from mock-infected cells. Comparative tryptic peptide analysis of the 13-kilodalton polypeptides produced in vivo and in vitro showed them to be identical. Thus, the s1 mRNA of reovirus type 3 is apparently bicistronic, and we suggest that the approximately 13-kilodalton polypeptide be called sigma s (standing for sigma small).
Preferential stimulation of rabbit alpha globin mRNA translation by a cap-binding protein complex.
A cap-binding protein complex (Edery et al. (1983) J. Biol. Chem. 258, 11398-11403) is shown here to stimulate preferentially the translation of endogenous alpha versus beta globin mRNA in a rabbit reticulocyte lysate. Several initiation factors (eIF-2, eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4E and eIF-5) and elongation factor 1 were found to have no such discriminatory effect. These results are in contrast to several previous reports and demonstrate that the only factor capable of relieving translational competition between alpha and beta globin mRNAs is the cap-binding protein complex.
Nucleic acid synthesis by isolated plant nuclei.
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