The goal continues to be to reach a molecular genetic understanding of the genome alternation process of Oxytricha fallax macronuclear (MAC) development: (1) how and what sequences are eliminated and how (2) the bodies and (3) telomeres of MAC minichromosomes are assembled. (1) Eliminated germline (micronuclear, MIC) sequences of two types will be studied. Internal eliminated sequences(IESs) interrupt sequences destined to be joined. Further IESs will be characterized and micro-injected IESs will be tested for proper excision. Entire families of repetitive MIC sequences are eliminated; the TBE1 transposon family is one such, and the unusual structure of TBE1s(Telomere-Bearing) suggests testable models which predict an interaction between the process of transposition, elimination, and MAC chromosome excision. Predictions will be tested. Further MIC TBE families will be characterized, and the notion that rare MAC-telomeric-repeat blocks internal in the MIC chromosomes have arisen by insertion of MAC chromosomes back into the germline, and that they are eliminated. (2) MAC telomeres are added to nascent MAC chromosomes by an unknown mechanism. To study it two indirect approaches will be to further characterize mature MAC telomeres and to study the homologous telomeres on the MIC chromosomes. MIC telomeres will be examined by in situ hybridization, by cloning and structure determination of both the cloned DNAs and features of the native telomeres, and by studying their apparent drift in length. Unusually long MAC telomeres will be characterized as found in bulk and as cloned, from total MAC DNA and from MAC rDNA of different-aged Oxytricha clones. (3) MAC chromosome bodies appear to be excised from the polytene chromatids. We have shown regular alternate excision patterns at two levels of resolution. At fine resolution excision sites exist in tight clusters. These sites will be located and possible consensus sequences identified. To understand the largescal alternate processing patterns which give MAC chromosome families with grossly different-sized members, the genetic organization and expression control of such families-including candidate histone gene chromosome families - will be characterized. Tools. A new and efficient cloning vector for native MAC chromosomes will be developed. The transient expression of E. coli Beta-galactoside and the Tn5 """"""""neo"""""""" gene will be studied from DNA constructs micro-injected into the MAC, and permanent MAC transformation to paromomycin resistance will be attempted. Irreversible somatic genome alterations are central to the human immune system. Other aspects of normal development may prove to be regulated by lineage-specific genomic sculpturing. Abnormal alterations are central to the genesis and progression of many forms of cancer.
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