Initiation and translation in vitro of mRNA for MOPC 315 immunoglobulin heavy chain and characterization of translation product.

An initiation study of mineral oil-induced plasmacytoma (MOPC) 315 heavy chain immunoglobulin (H315) in vitro has been conducted using formyl-[35S]methionyl-tRNAfMet and a highly purified 18 S message from MOPC 315 solid tumor in a crude rabbit reticulocyte lysate system. The product was specifically precipitated by antibodies directed against MOPC 315 immunoglobulin and H315. The in vitro H315 products terminally labeled with formyl-[35S]methionine or internally labeled with [3H]leucine were electrophoretically identical with in vivo H315 on sodium dodecyl sulfate-polyacrylamide gels. All of the [35S]-methionine was incorporated at the NH2 terminus, not internally, since there is a near complete recovery of [35S]methionine following one cycle of Edman degradation. The NH2-terminal cyanogen bromide peptide, CN2, of in vivo and in vitro H315 co-migrated exactly on gel electrophoresis under conditions which completely resolved two proteins differing in size by only 14 amino acids. These data strongly suggest that there is no NH2-terminal precursor of H315 in this system. Cyanogen bromide peptide profiles of in vivo and in vitro H315 were chromatographically indistinguishable. Three peptides, CN1, CN2, and CN4, which represent approximately 85% of the total amino acids of H315 were isolated and further characterized by electrophoresis and paper chromatography. All were very similar to the corresponding peptides of authentic H315. We conclude that the fidelity of H315 translation is preserved in vitro.     

Origins of immunoglobulin heavy chain domains

Summary Using computer programs that analyze the evolutionary history and probability of relationship of protein sequences, we have investigated the gene duplication events that led to the present configuration of immunoglobulin C regions, with particular attention to the origins of the homology regions (domains) of the heavy chains. We conclude that all of the sequenced heavy chains share a common ancestor consisting of four domains and that the two shorter heavy chains, alpha and gamma, have independently lost most of the second domain. These conclusions allow us to align corresponding regions of these sequences for the purpose of deriving evolutionary trees. Three independent internal gene duplications are postulated to explain the observed pattern of relationships among the four domains: first a duplication of the ancestral single domain C region, followed by independent duplications of the resulting first and last domains. In these studies there was no evidence of crossing-over and recombination between ancestral chains of different classes; however, certain types of recombinations would not be detectable from the available sequence data.     

The immunoglobulin heavy chain class switch

Conclusions While much has been learned concerning the molecular structural basis for the heavy chain class switch, many questions relating to the regulation of the switch remain unanswered, or at least controversial. Identification of the enzyme system which mediates the class switch, as well as other regulatory, possibly X-linked, genes should provide the necessary key to our understanding of this unique process.AbbreviationsB cell lymphocyte derived from the bone marrow in adult mammals or the bursa of Fabricius in chickens - bp base pair - C immunoglobulin constant region - CDR complementarity-determining region of the immunoglobulin variable region - D diversity gene segment of the immunoglobulin heavy chain variable region gene - H immunoglobulin heavy chain - Ig immunoglobulin - J joining region gene segment of the immunoglobulin variable region gene - kb kilobase - L immunoglobulin light chain - LPS lipopolysaccharide - Pyr pyrimidine - S-, s-site, s-region switch rearrangement site - SCE sister chromatid exchange - sIg surface immunoglobulin - T cell lymphocyte derived from the thymus - USCE unequal sister chromatid exchange - V immunoglobulin variable region     

Rat immunoglobulin E heavy chain locus

A 2100 base-pair long sequence has been established which covers all four constant domains of the rat epsilon-chain. An analysis of messenger RNA from an immunoglobulin E producing rat immunocytoma revealed two separate epsilon-chain mRNA species, 2.3 X 10(3) and 2.8 X 10(3) base-pairs long. The latter mRNA encodes the membrane binding form of the epsilon-chain. The membrane exons which are located approximately 2 X 10(3) base-pairs away from the 3'-side of the CH4 exon were also sequenced. A comparison between the rat and mouse epsilon-chains at the protein sequence level revealed an overall homology of 80% which, as expected, is considerably higher than the homology found between rat and human epsilon-chains. The fourth constant domain together with the two membrane exons exhibited the highest degree of homology, 81 to 89%. Only two differences were found when the epsilon-chains from LOU and Sprague Dawley rats were compared. The most striking difference at the nucleotide sequence level between the rat, mouse, and human epsilon genes was found within the first intron. The mouse genome contains a unique 366 base-pair long sequence in this region. The inserted sequence is repetitive and present in approximately 100 copies in the mouse genome. It is flanked by 22 base-pair long direct repeats and contains also 14 base-pair long inverted repeats, thus having properties in common with transposable elements.     

Gene transfer of immunoglobulin light chain restores heavy chain secretion

Several lines of evidence suggest that immunoglobulin (Ig) light (L) chain plays a role in the secretion of heavy (H) chain. For example, myeloma variant lines, which synthesize the Ig H chain but not the L chain, fail to secrete H chain protein. Here we have tested directly the role of chain assembly in the control of Ig secretion by the transfer of functional L chain genes into two such L chain-defective myeloma mutants. A lambda 2 or kappa L chain gene was introduced into variant lines of the mouse myelomas MOPC 315 (IgA, lambda 2) or PC7 (IgM, kappa), respectively. Although the two mutant lines are unable to secrete the H chain they produce, rescue of secretion of complete Ig protein molecules (IgA or IgM) was observed after transfection. These results imply that the secretory apparatus of these cells is intact and that the failure to secrete free H chain reflects a structural feature intrinsic to that protein. The implications of these results with respect to control of secretion of multi-subunit proteins are discussed.     

Purification and translation of an immunoglobulin lambda chain messenger RNA from mouse myeloma.

Here we describe the 500-fold purification of an mRNA encoding an immunoglobulin lambda light chain derived from the mouse myeloma tumor, RPC-20. Purification involves the isolation of membrane-bound polysomes, oligo(dT)-cellulose chromatography, and sucrose gradient centrifugation under conditions favoring denaturation of polynucleotide complexes. The mRNA purified in this way directs the cell-free synthesis of a polypeptide which is five or six amino acids longer than the mature form of RPC-20 light chain. In addition to directing the synthesis of a precursor-like polypeptide, the mRNA migrates on electrophoresis as a band containing approximately 1150 nucleotides, about 500 more than required to encode the mature form of the light chain.     

Sequence analysis of cloned cDNA encoding part of an immunoglobulin heavy chain.

The recombinant plasmid pH21-1 consists of mouse-derived complementary DNA (cDNA) in the E. coli plasmid pMB9. The mouse insertion has been completely sequenced, and encodes the CH3 domain and half the CH2 domain of the immunoglobulin gamma1 heavy chain. The predicted amino acid sequence differs at several positions from that previously published for this protein. The pattern of codon usage resembles that in some other eukaryotic messenger RNAs. A computer program has been used to predict the optimum secondary structure for the mRNA encoding the CH3 domain and the inter-domain junction.     

Primate immunoglobulin heavy chain constant gamma genes: an hypothesis of their evolution

Four Immunoglobulin gamma heavy chain isotypes are present in humans; the true phylogenetic relationship between the genes are not known because of the complex concerted evolution of the Ig multigene locus. Here we present data obtained from Southern blot analysis of the gamma genes in several primate species including prosimians (Lemur catta), New World Monkeys (Saguinus oedipus) and Old World Monkeys (Cercopitecus aethiops andMacaca fascicularis). Our data show the presence of a single IgG gene inLemur and probably inS.l oedipus, and of multiple genes in the two Cercopithecinae. These findings suggest that a single IgG gene was present in the ancestor of primates: we suppose that this IgG gene was later duplicated several times during primate evolution.     

Cytogenetic mapping of immunoglobulin heavy chain genes in Antarctic fish

The chromosomal location of the IgH locus has been analyzed in several bony fish of the Antarctic perciform group Notothenioidei. Two IgH probes were prepared from the species Trematomus bernacchii (family Nototheniidae, tribe Trematominae) and mapped onto the chromosomes of ten species belonging to the same genus (Trematomus) and in two outgroups, through one-color and two-color FISH. A single location of the IgH locus was found in the majority of the species examined, including the outgroups, whereas in four of them the IgH genes splited to two chromosomal loci. RT-PCR experiments revealed the presence of three allelic sequences in T. newnesi, a species in which the IgH genes were organized in two chromosomal loci. Possible pathways leading to IgH genes duplication during the diversification of trematomine fishes were inferred from the analysis of the FISH patterns in a phylogenetic context. The present work provides the first comprehensive picture of IgH genes organization at chromosomal level in a bony fish group.     

Ordered rearrangement of immunoglobulin heavy chain variable region segments

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.     

Allelic forms of the immunoglobulin heavy chain variable region

The complete variable region sequence of the heavy chain from a phosphorylcholine-binding myeloma protein of C57/BL allotype has been determined. When this sequence was compared with the germ line-coded heavy chain variable region sequence of BALB/c phosphorylcholine-binding proteins, five differences were observed. Four of the substitutions were located in the framework portion of the variable region and the fifth in the "J" or joining segment. Two of the framework substitutions were found at positions 14 and 16. Previous studies have shown that heavy chains from all anti-phosphorylcholine antibodies induced in C57/BL mice have the same amino acids at positions 14 and 16 as the C57/BL myeloma protein described in this communication. It has therefore been concluded that these residues are encoded in the C57/BL germ line in contrast to two alternatives in the BALB/c genome. This finding, in addition to the 96% homology found between the C57/BL and BALB/c sequences, suggests that these structures represent allelic forms of an entire variable region.     

Organization and polymorphism of rabbit immunoglobulin heavy chain genes

Germline genes encoding C mu, C gamma, C alpha, and C epsilon heavy chains of rabbit immunoglobulins have been isolated from recombinant phage and cosmid libraries. The JH, C mu, C gamma, and C epsilon are found in a 5'-JH-C mu-C gamma-C epsilon-3' orientation on a 90kb stretch of DNA. Four C alpha genes have been cloned and presumably reside 3' to the other CH genes. Southern blot analysis of rabbit sperm DNA indicates that the rabbit genome contains a single C gamma gene, one C mu gene, and as many as 10 C alpha genes. Restriction site polymorphism is found for C mu, C gamma, and C alpha genes of rabbits of various heavy chain haplotypes. The organization of the rabbit CH genes differs from that of mouse and human CH genes in that the rabbit has multiple C alpha genes, whereas mouse and human have one or two C alpha genes, respectively. In addition, mouse and human have four C gamma genes, whereas rabbit has only one C gamma gene. The presence of a single C gamma gene indicates that at least in the rabbits examined, no germline gene encoding latent or unexpected, C gamma allotypes is present. The genetic control of the expression of latent C gamma allotypes is discussed.     

Organization of human immunoglobulin heavy chain diversity gene loci

The variable region of immunoglobulin heavy chain is encoded by three separate genes on the germline genome: variable (VH), diversity (DH) and joining (JH) genes. Most human DH genes are encoded in 9-kb repeating sequences. We determined the nucleotide sequence of a 15-kb DNA fragment containing more than one and a half of these repeating units, and identified 12 different DH genes. Based on the sequence similarities of DH coding and the surrounding regions, they can be classified into six different DH gene families (DXP, DA, DK, DN, DM and DLR). Nucleotide sequences of DH genes belonging to different families diverge greatly, while those belonging to the same families are well conserved. Since the 9-kb DNA containing the six DH genes are multiplied at least five times, the total number of DH genes must be approximately 30. These DH genes are sandwiched by 12-nucleotide spacer signals. Most of the somatic DH sequences found in the published VH-DH-JH structures (the somatic DH segment being defined as the region which is not encoded either by germline VH or JH gene) were assigned to one of the germline DH genes. Other than these typical DH genes, however, we found a new kind of DH gene (which we termed DIR) the spacer lengths of whose neighbouring signals were irregular. The DIR gene appears to be involved in DIR-DH or DH-DIR joining by inversion or deletion. Two of the somatic DH sequences were assigned to the DIR genes. Long N segments might, therefore, originate from DIR genes.     

Biallelic heavy chain immunoglobulin gene rearrangement in acute nonlymphocytic leukemia

Acute nonlymphocytic leukemia (ANLL) was diagnosed in 18 patients based on morphological, cytochemical and immunological criteria. Leukemic cells of these cases were subjected to Southern blot analysis and subsequent hybridization to heavy chain immunoglobulin and T-cell-receptor gene probes. A rearrangement within the immunoglobulin joining region was detected in 2 cases, while the T-cell-receptor beta-chain gene was in germline configuration in all samples investigated. These data confirm recent reports indicating that immunoglobulin heavy chain gene rearrangements are not restricted to B-lineage neoplasms.