Michael Steinmetz

A pseudogene homologous to mouse transplantation antigens: Transplantation antigens are encoded by eight exons that correlate with protein domains

We have isolated about 30 to 40 different BALB/c mouse sperm DNA genomic clones that hybridize to cDNA clones encoding proteins homologous to transplantation antigens. One of these clones (27.1) was selected for sequence because it was polymorphic in Southern blot analysis of the DNAs from BALB/c and CBA mice. A fragment of 5.7 kilobases of this clone was completely sequenced and found to contain a pseudogene whose sequence is highly homologous to the sequences of known transplantation antigens. Pseudogene 27.1 is split into eight exons that correlate with the structurally defined protein domains of transplantation antigens. Using Southern blot hybridization on the DNAs of different inbred mouse strains, we mapped the pseudogene to the Qa-2,3 region, a part of the Tla complex on chromosome 17 that is adjacent to the major histocompatibility complex. The Qa2,3 region encodes lymphoid differentiation antigens homologous to the transplantation antigens in size, in peptide map profiles and in their association with beta2-microglobulin. These mapping studies suggest that gene 27.1 may be a pseudogene for eigher a Qa antigen or an as yet undefined transplantation antigen. Accordingly, we may have isolate genes encoding lymphoid differentiation antigens of the Tla complex as well as those encoding transplantation antigens among the 30 to 40 different genomic clones isolated from our sperm library.

Three cDNA clones encoding mouse transplantation antigens: Homology to immunoglobulin genes

We constructed cDNA libraries from poly(A)+ RNA isolated from cell lines of two different inbred strains of mice, and screened the libraries with a cDNA clone encoding a human transplantation antigen. Three cDNA clones were identified, sequenced and found to encode amino acid sequences highly homologous to portions of a known mouse transplantation antigen. Comparison of the cDNA sequences of mouse transplantation antigens with the constant region domains of the mouse immunoglobulin mu gene reveals a striking homology, which suggests that the two genes share a common ancestor. Antibody genes undergo DNA rearrangement during B cell differentiation that are correlated with their expression. In contrast, DNA blots with these cDNA probes suggest that the genes for the transplantation antigens are not rearranged in the genomes of liver or embryo cells, which express these antigens, as compared with sperm cells, which do not express these antigens. In Bam Hl-digested liver DNAs from different inbred strains of mice, 10-15 bands of hybridization were found. Accordingly, the genes encoding the transplantation antigens appear to constitute a multigene family with similar gene numbers in different mice.

A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs.

Significance Microtubules are dynamic protein filaments assembled from tubulin subunits, which play a key role for cell division. Ligands that target microtubules and affect their dynamics belong to the most successful classes of chemotherapeutic drugs against cancer by inhibiting cell proliferation. Here we have analyzed three structurally unrelated drugs that destabilize microtubules, using X-ray crystallography. The data reveal a new tubulin-binding site for these drugs, which renders their mechanism of action distinct from that of other types of microtubule assembly inhibitors. Similar key interactions with tubulin are observed for all three ligands, thus defining a common pharmacophore. Our results offer an opportunity for the rational design of potent tubulin modulators for the development of more efficient cancer therapies. The recent success of antibody–drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubule-destabilizing agents. Here, we determined the high-resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is approved by the US Food and Drug Administration (FDA) for the treatment of advanced breast cancer. We found that the drug binds to a site on β-tubulin that is distinct from the vinca domain and that blocks the formation of longitudinal tubulin interactions in microtubules. We also solved crystal structures of tubulin in complex with both a variant of rhizoxin and the phase 1 drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and that the structures revealed a common pharmacophore for the three ligands. Our results delineate a distinct molecular mechanism of action for the inhibition of microtubule assembly by clinically relevant agents. They further provide a structural basis for the rational design of potent microtubule-destabilizing agents, thus opening opportunities for the development of next-generation ADCs for the treatment of cancer.

Structural Basis of Microtubule Stabilization by Discodermolide.

Microtubule‐stabilizing agents (MSAs) are widely used in chemotherapy. Using X‐ray crystallography we elucidated the detailed binding modes of two potent MSAs, (+)‐discodermolide (DDM) and the DDM–paclitaxel hybrid KS‐1‐199‐32, in the taxane pocket of β‐tubulin. The two compounds bind in a very similar hairpin conformation, as previously observed in solution. However, they stabilize the M‐loop of β‐tubulin differently: KS‐1‐199‐32 induces an M‐loop helical conformation that is not observed for DDM. In the context of the microtubule structure, both MSAs connect the β‐tubulin helices H6 and H7 and loop S9–S10 with the M‐loop. This is similar to the structural effects elicited by epothilone A, but distinct from paclitaxel. Together, our data reveal differential binding mechanisms of DDM and KS‐1‐199‐32 on tubulin.

Molecular immunology genes of the major histocompatibility complex of the mouse

Publisher Summary This chapter presents the current knowledge of genes of the major histocompatibility complex (MHC) of the mouse. The genes of the MHC of the mouse have been more intensely studied than those of other vertebrates mainly because of the availability of a large number of inbred, thoroughly characterized congenic and recombinant congenic strains. The MHC is a cluster of tightly linked genes coding for molecules involved in the immune response. Three classes of genes have been identified. Class I genes encode the classical transplantation antigens and a number of hematopoietic differentiation antigens, structurally closely related to the transplantation antigens. Class II genes encode the Ia or immune response-associated antigens and class III genes encode complement components. The chapter focuses on the functional and structural studies related to these class of genes of BALB/c mouse. It is found that over 1000 kb of DNA from the MHC of the BALB/c mouse has been cloned and characterized.

Transgenic Mice for Analysis of T Cell Development

Publisher Summary This chapter presents an analysis of T-cell development in mice. The complexity of the immune system has its molecular basis in the enormous diversity of T-cell receptors (TCRs) and immunoglobulin (Ig) structures. Genes coding for the α and β chains of the TCR are assembled from variable (V), diversity (D), and joining (J) gene segments during thymocyte development when the β locus rearranges first. The functional rearrangement on one chromosome blocks further rearrangement of the other allele, known as allelic exclusion, and leads to a clonal distribution of different α and β chains. Combinatorial joining of different V, D, and J gene segments for α and β chains, together with N region diversity and combinatorial association of α and β chains, allows the generation of up to 10 20 different TCR specificities. Although the use of a selectable marker gene usually results in the disruption of the target gene, more subtle mutations are also possible.

Isolation and Characterization of I Region Genes from the Major Histocompatibility Complex of the Mouse

The major histocompatibility complex on chromosome 17 of the mouse contains several families of genes which encode cell-surface recognition structures (1). One of these families, the immune response genes of the I region, is of particular interest because it regulates the ability of mice to make immune responses to simple antigens. A question of fundamental importance is how the immune response genes function. One hypothesis is that they are the genes encoding the T-cell receptors (2). More recent data suggest that the immune response gene products are in fact the Ia antigens, cell-surface molecules found on B cells, macrophages, and some T cells which have been analyzed serologically (3).