Stephen Crews

A single VH gene segment encodes the immune response to phosphorylcholine: Somatic mutation is correlated with the class of the antibody

Abstract The immune response in BALB/c mice to phosphorylcholine is highly restricted in its heterogeneity. Of the 19 immunoglobulins binding phosphorylcholine for which complete V H -segment amino acid sequences have been determined, 10 employ a single sequence, denoted T15 after the prototype V H sequence of this group of antibodies. The remaining 9 of these V H segments are variants differing by 1 to 8 residues from the T15 sequence. Using a cloned V H cDNA probe complementary to the T15 sequence, we isolated from a mouse sperm genomic library clones corresponding to four V H gene segments that by DNA sequence analysis are >85% homologous to one another. These four V H gene segments have been denoted the T15 V H gene family. These V H gene segments are most, if not all, of the germline V H gene segments that could encode the V H sequences of antibodies that bind phosphorylcholine. One of these four genes contains the T15-V H -coding sequence. When the T15-family V H gene segments were compared with the complete V H protein sequences of 19 hybridoma and myeloma immunoglobulins that bind phosphorylcholine, several striking conclusions could be drawn. First, all of these V H regions must have arisen from the germline T15 V H gene segment. Thus virtually the entire immune response to phosphorylcholine is derived from a single V H -coding sequence. Nine of the 19 V H regions were variants differing from the T15-V H -coding sequence and, accordingly, must have arisen by a mechanism of somatic diversification. Second, the variants appear to be generated by a somatic mutation mechanism. They cannot be explained by recombination or gene conversion among members of the T15 gene family. Third, somatic mutation is correlated with the class of the antibody. All of the somatic variation is found in the V H regions derived from antibodies of the IgA and IgG classes. The IgM molecules express the germline T15 V H gene segment exclusively.

The Generation of Diversity in Phosphorylcholine-Binding Antibodies

Publisher Summary Antibody heavy and light chains are encoded by more than one gene, thus anticipating the noncontiguous nature of eukaryotic genes and the DNA rearrangements that are central to the formation of antibody coding regions. Detailed structural analysis revealed that the amino terminal variable regions of both heavy and light chains contain three short segments of hypervariability. These hypervariable regions were shown by X-ray crystallography to comprise the antibody-combining site, whereas the remaining portions of the variable region are relatively invariant in structure and hence are called “framework” regions. The structural analysis of antibody genes was achieved for the most part using murine myeloma tumors, generally induced by intraperitoneal administration of mineral oil, as a source of cells “frozen” at the level of plasma cell differentiation. The murine antibodies directed against phosphorylcholine (PC) discussed in this chapter proved an ideal system for delineating the fundamental mechanisms that generate the immunoglobulin repertoire of higher vertebrates. This family utilizes all of the described mechanisms that contribute to antibody heterogeneity. Structural analysis of the anti-PC antibodies and the genes that encode them illuminates the multiple strategies utilized by higher vertebrates to amplify a limited amount of genetic information to permit the expression of at least 10 million different antibodies. Of the thousands of different germline antibody gene segments, fewer than a dozen are employed in PC-binding antibodies.

The sequences of the small ribosomal RNA gene and the phenylalanine tRNA gene are joined end to end in human mitochondrial DNA

Summary The 5′ end proximal regions of the two HeLa cell mitochondrial rRNAs (16S and 12S) have been sequenced by partial enzymatic digestions of the 5′ end 32 P-labeled RNAs followed by electrophoretic fractionation of the products on polyacrylamide/urea gels. Likewise, a 600 nucleotide mitochondrial DNA (mit-DNA) fragment, previously designated Δ8a Hae , that contains the 5′ end of the 12S rRNA, has been sequenced by the method of Maxam and Gilbert. The first 71 nucleotides of the 12S rRNA and the DNA coding sequence have been aligned and found to be colinear. This observation extends to the 5′ end proximal segment of the 12S rRNA gene the conclusion of earlier experiments, indicating the absence of intervening sequences in the body of the small rRNA gene. A comparison of the 12S rRNA coding sequence determined here (286 nucleotides) with that of an E. coli 16S rRNA gene has revealed significant homologies. Previous electron microscopic analysis of hybrids between the heavy (H) strand of mit-DNA and ferritin-labeled mitochondrial 4S RNAs had shown the presence of a 4S RNA gene near the 5′ end of the 12S rRNA coding sequence. In the present work, a search of the DNA sequence for a cloverleaf structure has indeed revealed the occurrence of a tRNA Phe gene. The unexpected finding, however, has been that the 3′ end of this gene is contiguous to the 5′ end of the 12S rRNA coding sequence without any intervening nucleotides.

A small polyadenylated RNA (7 S RNA), containing a putative ribosome attachment site, maps near the origin of human mitochondrial DNA replication

Abstract The light (L) strand sequence of HeLa cell mitochondrial DNA which codes for a small polyadenylated RNA (7 S RNA) has been precisely localized, by mapping and sequencing studies, in a region of the genome which immediately precedes the origin of replication in the direction of L-strand transcription, extending from 219 nucleotides to within 20 nucleotides from this origin. A 5′-end sequencing analysis of 7 S RNA has revealed the presence of two major species differing in size by one nucleotide at this end, and possibly three minor species. The 7 S RNA appears to contain, near its 3′-end, a potential reading frame for a polypeptide 23 or 24 amino acids long. Furthermore, in the 5′ non-coding region, an 11-nucleotide long sequence has been identified which is complementary to a sequence near the 3′-end of the 12 S ribosomal RNA, and possibly represents a ribosome attachment site.

A Comprehensive View of Mitochondrial Gene Expression in Human Cells

In the past 2 years, the determination of the complete sequence of the human and bovine mtDNAs by Sanger’s group in Cambridge, England (see Anderson et al., this volume) has revealed the unique features of the genetic code and of the gene organization in the mammalian mitochondrial genome (Anderson et al. 1981). The parallel work carried out in our laboratory on the organization of the mtDNA transcripts in HeLa cells has produced a perfectly matching picture, providing at the same time important information on both the structure of the mitochondrial genes and their mode of expression (Ojala et al. 1980Ojala et al. 1981; Montoya et al. 1981). Thus, the extraordinarily compact gene organization of the mammalian mtDNA, with its continuous genes mostly butt-jointed to each other and a nearly complete absence of noncoding stretches, has been shown to have its precise counterpart in the tight arrangement of the mtDNA transcripts and in the distinctive structural features of the mitochondrial mRNAs. Moreover, the interspersion of the tRNA genes with the rRNA and protein-coding genes and their immediate juxtaposition have pointed to an additional functional role for tRNA sequences related to their positions in mtDNA, namely, as recognition signals for RNA processing (Attardi et al. 1980a; Ojala et al. 1981). The information on the structural organization of the genes and their transcripts in the human mitochondrial genome has further provided the framework for an in-depth analysis of the metabolic behavior of mtRNAs in HeLa cells (Gelfand and Attardi 1981). The correlation and integration of...

THE ORGANIZATION OF THE GENES IN THE HUMAN MITOCHONDRIAL GENOME AND THEIR MODE OF TRANSCRIPTION

ABSTRACT The organization of the genes in the human mitochondrial genome and their mode of transcription are being investigated by a detailed mapping, structural and metabolic analysis of the various discrete poly(A)-containing and non-poly(A)-containing RNA species coded for by mitochondrial DNA (mit-DNA). Several approaches, involving different RNA-DNA hybridization techniques, RNA and DNA sequencing methods, and analysis of kinetic behavior, are being used in this work. A fine structural study of the ribosomal RNA region of mit-DNA has failed to reveal the presence of inserts in the main body of the 12S and 16S rRNA cistrons. The sequence of 71 nucleotides at the 5′-end of 12S rRNA and that of 32 nucleotides at the 5′-end of 16S rRNA have been determined. Experiments of hybridization of nascent RNA chains with different restriction fragments of mitochondrial DNA have led to the identification in this DNA of a region containing a point of initiation of L strand transcription. Several giant poly(A)-containing and non-poly(A)-containing L strand transcripts have been identified and characterized in their map location and metabolic behavior.