In the genome of a germ-line cell, the genetic information for an immunoglobulin polypeptide chain is contained in multiple gene segments scattered along a chromosome. During the development of bone marrow-derived lymphocytes, these gene segments are assembled by recombination which leads to the formation of a complete gene. In addition, mutations are somatically introduced at a high rate into the amino-terminal region. Both somatic recombination and mutation contribute greatly to an increase in the diversity of antibody synthesized by a single organism.

Lipases belong to a class of esterases whose activity on triglycerides is greatly enhanced at lipid-water interfaces. This phenomenon, called interfacial activation, has a structural explanation: a hydrophobic lid, which at rest covers the catalytic site, is displaced on substrate or inhibitor binding and probably interacts with the lipid matrix. Fusarium solani pisi cutinase belongs to a group of homologous enzymes of relative molecular mass 22-25K (ref. 7) capable of degrading cutin, the insoluble lipid-polyester matrix covering the surface of plants, and hydrolysing triglycerides. Cutinases differ from classical lipases in that they do not exhibit interfacial activation; they are active on soluble as well as on emulsified triglycerides. Cutinases therefore establish a bridge between esterases and lipases. We report here the three-dimensional structure of a recombinant cutinase from F. solani pisi, expressed in Escherichia coli. Cutinase is an alpha-beta protein; the active site is composed of the triad Ser 120, His 188 and Asp 175. Unlike other lipases, the catalytic serine is not buried under surface loops, but is accessible to solvent. This could explain why cutinase does not display interfacial activation.

The nuclear RNA from a large variety of K-producing plasmacytomas was size fractionated and analyzed with a series of cloned probes representing sequences encoding variable (V), joining (J), and constant (C) regions and selected intervening sequences. All of the plasmacytomas produce a nuclear RNA component that contains V, and C, sequences as well as the intervening sequence between Jo and C,, and that has a distinctive size depending on which of the four Jo segments is expressed (i.e., is present in the secreted K chain). These RNAs are the precursors of K mRNAs, which are transcribed from productively rearranged C, genes. Half of the plasmacytomas examined produce, in addition to a K mRNA precursor, a discrete component of about 8.4 kilobases that contains C, and upstream flanking sequences but lacks the expressed V region sequence.The ability to produce this component is always associated with the persistence in the tumor genome of an unrearranged (germline) J,,-C region. In tumors rearranged at both K loci the nonproductive allele is either transcriptionally silent or, in a minority of cases, transcribed and processed into a "fragment" mRNA lacking V region sequences. These results reveal that allelic exclusion can be effected at several levels of gene expression. They also provide some insight into the relative contributions of the V and C gene elements to this expression. The formation of an active immunoglobulin gene requires a reorganization of the germline genome to bring the elements coding for the variable (V) and constant (C) regions within a single transcriptional unit (1). In the case of mouse K chains this reorganization appears to involve a deletion of the DNA that separates the expressed VK gene and any one of four different JK (joining) gene segments that are located from 2.7 (J4) to 3.9 kilobases (kb) (J1) upstream from the CK gene § (2, 3). Transcription of such a rearranged K locus results in the formation of a pre-mRNA that contains the intervening sequence between the CK gene and the expressed JI segment (J-C intron) (4-7). This intron is subsequently excised during the processing of the K mRNA. Because the length of the J-C intron is different for each of the four IK segments, plasmacytomas expressing different JK segments contain pre-mRNAs of characteristic and distinctive sizes (7). The observation that productive rearrangements are found on only one of the two chromosomes bearing the K genes is thought to provide a molecular basis for the phenomenon of allelic exclusion (1, 8). However, there is presently some uncertainty about the nature and extent of rearrangements on the chromosome bearing the "nonexpressed" CK allele (8-i1). Moreover, the relationship between the structural organization of the K locus and its transcriptional activity is also obscure.In several plasmacytomas a large (n-9 kb) CK-containing transcript is produced, which, in contrast to the pre-mRNAs described above, has a uniform size irrespective of which JK segment is expressed (7). We initially thought th...

Site-directed mutagenesis in the active site of xylose isomerase derived from Actinoplanes missouriensis is used to investigate the structural and functional role of specific residues. The mutagenesis work together with the crystallographic studies presented in detail in two accompanying papers adds significantly to the understanding of the catalytic mechanism of this enzyme. Changes caused by introduced mutations emphasize the correlation between substrate specificity and cation preference. Mutations in both His 220 and His 54 mainly affect the catalytic rate constant, with catalysis being severely reduced but not abolished, suggesting that both histidines are important, but not essential, for catalysis. Our results thus challenge the hypothesis that His 54 acts as an obligatory catalytic base for ring opening; this residue appears instead to be implicated in governing the anomeric specificity. With none of the active site histidines acting as a catalytic base, the role of the cations in catalyzing proton transfer is confirmed. In addition, Lys 183 appears to play a crucial part in the isomerization step, by assisting the proton shuttle. Other residues also are important but to a lesser extent. The conserved Lys 294 is indirectly involved in binding the activating cations. Among the active site aromatic residues, the tryptophans (16 and 137) play a role in maintaining the general architecture of the substrate binding site while the role of Phe 26 seems to be purely structural.

Several genes for the variable region of immunoglobulin heavy chains (VH genes) have been isolated from human fetal liver DNA by using a cDNA plasmid probe containing a mouse VH sequence. The detectable VH genes are separated by [12][13][14][15][16] kilobases of DNA, and hybridization experiments show about 23 hybridizing VH genes in DNA of three different individuals. The complete nucleotide sequence of one of these human VH genes shows that it belongs to the human VHIII subgroup. The VH gene appears to contain an intervening sequence (104 bases in length) within a precursor sequence, between residues -4 and'-5. The precursor sequence is itself 19 codons in length. The 3' end ofthe V gene seems to be at codon 93 or 94, and this is followed by the conserved sequences C-A-C-A-G-T-G and G-A-CA--A-A-A-C-C. The presence of these sequences suggests that similar enzymatic mechanisms are involved in the integration of V genes in both heavy and light chains. Immunoglobulins are made of two types of polypeptide chain, the heavy (H) and the light (L) chains, and each of these chains has an NH2-terminal variable (V) region covalently attached to a COOH-terminal constant (C) region. The variability in the sequence of the V regions of antibodies is the feature that gives rise to the diversity of the antibody response, and an outstanding problem is the extent of inherited V gene diversity compared to additional diversity that may be generated by somatic means. In addition, as predicted by the two-genes-one-polypeptide hypothesis (1), the V genes are inherited as a separate set of genes that are linked to the respective C genes (2). In mouse K light chains it is clear that a set of VK genes exists as a separate unit from a CK gene; integration of one of the VK genes and the CK gene results in the functional K chain gene (3)(4)(5). Nucleotide sequence data of VX (6) and VK (7-9) L chains has shown that the V gene sequence terminates at the codon for amino acid 96. The rest of the V region gene comes from one of a set of joining segments (JL). After VL-JL integration the fused V gene remains separated from the C gene by a large intervening sequence (4, 7-9). A further intervening sequence occurs within the precursor region of both VA and VK genes, and all these sequences are transcribed and subsequently removed from the nuclear RNA (10,11 MATERIALS AND METHODSThe human fetal liver phage library was kindly given by T. Maniatis and was constructed by Hae III/AIu I digestion of DNA followed by the addition of EcoRI linker molecules and insertion into X Charon 4A (12). Plaque hybridization (13) of the library was carried out after plating on Nunc bioassay plates (23 X 23 cm), using the nick-translated plasmid pg/107 (14).Restriction endonuclease mapping of purified positive phage was carried out by using single and double digests of EcoRI, Kpn I, Bgl II, Bst I, and Xho I, comparing sizes to those of the X Charon 4A host (15), hybridizing, and determining the nucleotide sequence. Southern filter hybridization analysis (16) ...