Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis.

Cdc16p, Cdc23p and Cdc27p are all essential proteins required for cell cycle progression through mitosis in Saccharomyces cerevisiae. All three proteins contain multiple tandemly repeated 34 amino acid tetratricopeptide repeats (TPRs). Using two independent assays, two-hybrid analysis in vivo and co-immunoprecipitation in vitro, we demonstrate that Cdc16p, Cdc23p and Cdc27p self associate and interact with one another to form a macromolecular complex. A temperature sensitive mutation in the most highly conserved TPR domain of Cdc27p results in a greatly reduced ability to interact with Cdc23p, but has no effect on interactions with wild-type Cdc27p or Cdc16p. The specificity of this effect indicates that TPRs can mediate protein-protein interactions and that this mutation may define an essential interaction for cell cycle progression in yeast. The conservation of at least two of the three proteins from yeast to man suggests that this protein complex is essential for mitosis in a wide range of eukaryotes.

Construction of human chromosome 21-specific yeast artificial chromosomes.

Chromosome 21-specific yeast artificial chromosomes (YACs) have been constructed by a method that performs all steps in agarose, allowing size selection by pulsed-field gel electrophoresis and the use of nanogram to microgram quantities of DNA. The DNA sources used were hybrid cell line WAV-17, containing chromosome 21 as the only human chromosome and flow-sorted chromosome 21. The transformation efficiency of ligation products was similar to that obtained in aqueous transformations and yielded YACs with sizes ranging from 100 kilobases (kb) to greater than 1 megabase when polyamines were included in the transformation procedure. Twenty-five YACs containing human DNA have been obtained from a mouse-human hybrid, ranging in size from 200 to greater than 1000 kb, with an average size of 410 kb. Ten of these YACs were localized to subregions of chromosome 21 by hybridization of RNA probes (corresponding to the YAC ends recovered in Escherichia coli) to a panel of somatic cell hybrid DNA. Twenty-one human YACs, ranging in size from 100 to 500 kb, with an average size of 150 kb, were obtained from approximately equal to 50 ng of flow-sorted chromosome 21 DNA. Three were localized to subregions of chromosome 21. YACs will aid the construction of a physical map of human chromosome 21 and the study of disorders associated with chromosome 21 such as Alzheimer disease and Down syndrome.

Identification of three new Ig lambda-like genes in man.

Three new human lambda L chain-like Ig genes are identified by restriction enzyme and nucleotide sequence analysis. Two genes, 14.1 and 16.1, have intact J and C regions, and are potentially functional, with open reading frames. A third gene, 18.1, is a pseudogene. The evolutionary lineage of these genes compared to the known functional locus lambda C1-lambda C6 can be surmised from Southern blot and nucleotide homologies. This study demonstrates that the human lambda gene family is more complex than previously recognized.

Variable amplification of immunoglobulin lambda light-chain genes in human populations.

The human lambda immunoglobulin locus displays a series of restriction fragment length polymorphisms that are readily detected in small populations of normal individuals. Similar polymorphisms appear in populations of wild mice, suggesting that the lambda locus is subject to rapid variation within a single species. Here we show that the polymorphisms seen in the human lambda locus seem to have arisen from unequal meiotic crossing over, altering the number of lambda from as few as six to as many as nine per haploid genome. This expansion and contraction in the number of human lambda genes is significant in that it may affect an individual's capacity to produce variation among lambda light chain genes.

Regulation of the humoral immune response: from immunoglobulin genes to regulatory T cell networks.

Leukemic lymphocytes were used in studies to define the rearrangements of immunoglobulin genes and the networks of immunoregulatory T cells that are important to the control of immunoglobulin synthesis in humans. Leukemic B cells displayed rearrangements of their expressed mu and k or lambda genes. Surprisingly, the k constant region genes were deleted or rearranged in all lambda-expressing B lymphocytes examined. Most non-T, non-B lymphocytic leukemias had immunoglobulin gene rearrangements, which indicates that these cells were committed to B cell development at the immunoglobulin gene level. The pattern of immunoglobulin gene rearrangements in the B cell precursor and B cell leukemias suggests an ordered hierarchy of somatic rearrangements with mu genes preceding light chains and k light chain genes preceding lambda. Some of the leukemic T cells studied were homogeneous populations of cells that retained immunoregulatory functions with many Sézary T leukemic cells functioning as helper cells in their interactions with B lymphocytes. In contrast, malignant cells from patients with the adult T leukemia of Japan frequently functioned as suppressor cells. In contrast to normal unactivated T cells or other leukemic T cells, the leukemic T cells from all adult T cell leukemia patients studied had an activated receptor for T cell growth factor. The malignant cells of a patient with an acute T cell leukemia functioned as prosuppressor cells. Under the influence of normal T cells or their secreted inducer products, these leukemic cells matured both functionally and in terms of cell surface antigens into suppressor effector cells.

Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins.

We have explored the relationship among immunoglobulin gene rearrangement, cytoplasmic immunoglobulin production, and cell surface antigen expression within 37 cases of acute lymphocytic leukemia. All 12 cases of the T cell type had germ-line kappa and lambda genes and 11 of 12 had germ-line heavy chain genes. In contrast, all 25 cases of the "non-T, non-B" classification, which lacked both definitive T cell markers and surface immunoglobulin, had rearranged immunoglobulin genes, indicating that they represent precursor cells already committed to the B cell lineage at the gene level. 14 had rearranged heavy chain genes, yet retained germ-line light chain genes, whereas 11 cases had both heavy and light chain gene reorganizations. All patterns of immunoglobulin gene rearrangement predicted by a model that proceeds from heavy chain gene recombination to light chain genes were observed. Despite the uniform presence of rearranged immunoglobulin genes, only five cases produced cytoplasmic mu-chain, one exceptional case produced gamma-chain, and another produced only lambda-chain. The cases of B cell precursor type that do not produce immunoglobulin may represent cells that frequently possess ineffectively rearranged immunoglobulin genes. Included in this group may be a set of cells trapped within the B cell precursor series because their ineffective rearrangements have eliminated certain gene subsegments necessary for the assemblage of an effective heavy chain gene. All seven cases of the non-T, non-B subgroup that bore HLA-DR but lacked CALLA (the common acute lymphocytic leukemia-associated antigen) represented the earliest recognizable stage of B cell precursors with rearranged heavy chain genes but germ-line light chain genes. Correlations here suggest that cells entering B cell development express HLA-DR and rearrange heavy chain genes before the expression of a B cell-associated antigen recognized by the antibody BA-1, the antigen CALLA, and any subsequent light chain gene rearrangements.

Normal human B cells display ordered light chain gene rearrangements and deletions.

Human kappa-producing B cell lines and leukemias retain their excluded lambda light chain genes in the germ line configuration, whereas transformed lambda-producing B cells uniformly rearrange or delete their kappa genes (12). Whether the unexpected lambda gene recombinations within malignant lambda-producing B cells reflect a normal developmental process or are secondary to transformation and specific chromosomal translocations was uncertain. To resolve this issue, we purified circulating lambda-bearing B cells from a normal individual to 97% purity by using a series of negative selection steps and a final positive selection on a cell sorter. Over 95% of the collective kappa genes in these lambda B cells were no longer in their germ line form, with the majority (60%) deleted and the remainder present but in a rearranged state. The chromosomal loss of the germ line kappa genes included the joining (J kappa) segments as well as the constant (C kappa) region, yet the particular variable (V kappa) gene family studied was spared. In addition, the incidence of kappa gene deletions was higher in long-term than in freshly transformed lambda B cell lines. This implies that the deletion of aberrantly rearranged kappa genes may occur as a second event. Such a mechanism would serve to eliminate aberrant transcripts and light chain fragments that might interfere with the synthesis and assembly of effective immunoglobulin molecules. Thus, despite the nearly equal usage of kappa and lambda light chain genes in man, there appears to be a sequential order to their expression during normal B cell ontogeny in which kappa gene rearrangements precede those of lambda.

Chromosomal location of human kappa and lambda immunoglobulin light chain constant region genes.

The chromosomal location of human constant region light chain immunoglobulin (Ig) genes has been determined by analyzing a group of human fibroblast/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human kappa and lambda constant region genes. Human chromosomes in each cell line were identified by isoenzyme analysis. The DNA from hybrid cells was digested with restriction endonucleases, size fractionated by gel electrophoresis, transferred to nitrocellulose or DBM paper, and hybridized with (32)P-labeled nucleic acid probes. The C(kappa) gene was assigned to human chromosome 2 and the C(lambda) genes to chromosome 22, based upon analysis of these hybrid cell lines, and these assignments were confirmed by analysis of subclones. A group of previously unassigned loci can be mapped to chromosome 2 by virtue of their close linkage to C(kappa). The lambda and kappa light chain and heavy chain Ig genes have now been assigned to all three human chromosomes that are involved in translocations with chromosome 8 in human B cell neoplasms. These techniques and probes provide a means to study the detailed arrangement of human Ig genes and their pseudogenes.

Evolution of human immunoglobulin kappa J region genes.

Immunoglobulin kappa chain variable region genes are assembled from two discontinuous DNA segments, a V and a J gene. The J region genes, in addition to encoding amino acid positions 96-108 of the kappa polypeptide chain, also provide sequences required for both DNA and RNA splicing reactions. For purposes of evolutionary comparison and to establish the complexity of the kappa J region locus in man, we have determined an approximately 3000 basepair nucleotide sequence in a cloned human DNA fragment that encodes the germline distinct J region segments. Significant blocks of homology have been tightly maintained between this region and an analogous segment of the mouse genome. In particular, the short sequences, GGTTTTTGT and CACTGTG, thought to be involved in V-J recombination, are the most highly conserved regions (97% homology). In addition, from heteroduplex data and computer analysis of the nucleotide sequences, it is clear that the mouse J3 sequence, a pseudogene, is not present in the human cluster. This can be explained by a duplication event in the mouse J region gene cluster that may have been the result of unequal crossing over between homologous chromosomes.

Clustered arrangement of immunoglobulin lambda constant region genes in man.

The immunoglobulin lambda light chain locus of man contains six lambda-like genes arranged tandemly on a 50-kilobase segment of chromosomal DNA. THe sequences of three of these genes correspond to three known non-allelic lambda chain isotypes: Mcg, Ke-Oz- and Ke-Oz+. They surround a highly polymorphic and evidently unstable region that is repeatedly deleted when cloned in Escherichia coli. Three additional, but as yet unlinked, lambda-like sequences have also been cloned, suggesting that the lambda genes form an unexpectedly large family within the human genome.

Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells.

We have used a special class of human acute lymphocytic leukemias, the common "non-T/non-B" cell type, to define a hierarchy of genetic rearrangements that occur during the earliest stages of B-cell maturation. This has allowed us to identify intermediate cells predicted by a hierarchial model in which immunoglobulin heavy chain variable region gene formation precedes that of light chain and in which kappa light chain gene formation precedes that of lambda. The model emphasizes the flexible nature of immunoglobulin gene recombination that not infrequently produces aberrant or null genes that are phenotypically excluded from expression. Remaining alleles or isotypic genes can then be utilized as "spares" undergoing recombination until a valid gene is formed. Significantly, the excluded allele or isotype is frequently deleted from the genome. In addition to defining a pathway of genetic maturation, this analysis provides a powerful means to further classify cases of non-T/non-B-cell acute lymphocytic leukemia, most of which seem to reside at early stages along the B-cell pathway of differentiation.

Human immunoglobulin kappa light-chain genes are deleted or rearranged in lambda-producing B cells.

We have examined 10 human lambda light-chain-producing B lymphocytes and found that genes for the kappa constant region have been deleted in each. Only one of the 20 possible kappa alleles in these cells has been retained and even this exception is rearranged. In contrast, lambda constant region genes remain in the germ-line configuration in each of the eight kappa light-chain-producing human B lymphocytes examined. Such observations suggest a hierarchy of light-chain-gene rearrangement beginning with kappa and proceeding to lambda.

Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments.

The human immune system offers special advantages for study of the development and evolution of the immune response. A variety of human cell lines are available that are arrested at various stages of development, and human genes provide a convenient evolutionary point of comparison with the already well characterized genes of the mouse. In this paper, we describe the procedure we have used to clone the human kappa chain genes in both germline and rearranged configurations. We have taken advantage of distantly related probes derived from the mouse and nonstringent conditions of hybridization to find the human genes among phage lambda recombinants formed with partially purified genomic restriction fragments of human DNA. In addition to establishing a physical map of the human kappa C and J regions, we have determined the entire sequence of a germline human constant region gene (the Inv3 allele) and two of its J segments, as well as the V/J recombination site of an active human kappa chain gene. For purposes of comparison, we also determined the sequence of the chromosomal mouse constant region gene and its flanking sequences. Although mouse and human sequences have changed extensively during the 70 million years since the two species diverged. significant blocks of homology have been conserved selectively. Some of these have obvious significance in terms of DNA and RNA splicing reactions. By forming heteroduplex structures between mouse and human genes we were able to identify four human J regions that are much more stringently conserved throughout their coding sequences than are the C region genes. In addition, the middle j structure (J3) of the mouse (which is thought to be inactive) appears to be missing from the human genome.

Identification of a cytidine-specific ribonuclease from chicken liver.

Rapid RNA sequencing technology was used to determine if the base specificities of an RNase recently purified from chicken liver would prove useful for RNA sequence analysis. Escherichia coli 5 S [5'-32P]rRNA or yeast 5.8 S [5'-32P]rRNA was digested with the enzyme and this digest, along with digests derived from RNases of known specificity (U2, T1, T2) were subjected to electrophoresis through denaturing polyacrylamide slab gels. Following autoradiography, the banding patterns arising from the activity of each enzyme were compared, and the base specificity of the unknown RNase was established. The chicken liver RNase was found to have a marked preference for phosphodiester bonds containing cytidylic acid residues, a property which should make the enzyme useful for distinguishing between pyrimidines in RNA sequencing.

Histone gene switch in the sea urchin embryo. Identification of late embryonic histone messenger ribonucleic acids and the control of their synthesis.

During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these unit. Four bands in the early mRNA sample are identified as H1, H3, H2A " H2B, and H4 mRNA while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both H3 and H2A mRNA. When a polysomal RNA preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A + H3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal RNA present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic RNA from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.

Stabilization of an RNA molecule by 3'-terminal poly (A)-induced inhibition of RNase activity.

The inhibitory properties of poly(A) on human spleen ribonuclease have been investigated. Hydrolytic activity has been shown to be strongly inhibited by poly(A) contained within RNAs isolated from a variety of natural sources. Furthermore, poly(A) segments of varying length have been covalently linked at the 3' terminus of Escherichia coli 5 S rRNA by polynucleotide phosphorylase in an attempt to construct an in vitro demonstration of the stabilization of RNA which contains poly(A). The extent to which these poly(A) tracts, varying from 4 to 132 nucleotides in length, could inhibit endonucleolytic attack on the 5 S rRNA to which they are linked was found to be dependent upon their length and upon small changes in spermidine concentration. The consequences of these findings are discussed in terms of a possible role for poly(A).