ABSTRACT

Abstract. cDNA clones encoding the a chain of the murine lymphocyte-Peyer's patch adhesion molecule (LPAM), which is associated with lymphocyte homing, have been isolated by screening with the human VLA-4 (a4h) probe . Several a4 antigenic determinants were identified on COS-7 cells after transfection . From overlapping clones, ti5 kb of contiguous nucleotide sequence have been determined, encoding a protein sequence of 1039 amino acids for the LPAM a chain (ON.). LPAM is a member of the integrin family of cell-surface heterodimers, and a4m is the murine homologue of the human 014h chain . The two proteins have a total sequence similarity of 84%, with an almost perfect conservation (31/32 amino acids) in the cytoplasmic domain . Like a4h, a4m is distinct from other integrin a chains because it has neither an I-domain nor a COON-terminal cleavage site. The positions of the critical step for the onset of an immune reaction resides in the ability of lymphocytes to cross blood vascular endothelium in order to enter various lym phoid organs, thus, recirculating between blood and lymph .

An rDNA size class in the genome of the nematode Ascaris lumbricoides is described which is interrupted by a 4.5‐kb long intervening sequence located in the 26S coding region. This molecular form occurs in approximately 15 copies per haploid genome and amounts to approximately 5% of the total nuclear rDNA. Intervening sequences are present only in the 8.8‐kb rDNA, but not in the 8.4‐kb rDNA repeating units of A. lumbricoides. Cloning of the interrupted rDNA units revealed, in addition to the main 4.5‐kb insertion, shorter intervening sequences of 4‐kb and 119‐bp length. Both shorter rDNA forms are present in the single copy range of the haploid genome. Sequence analyses of the intervening sequence/rDNA junctions show an identical right‐hand junction for all of the three different rDNA forms. The two shorter intervening sequences are a coterminal subset of the right‐hand end of the main 4.5‐kb insertion, whereas all three insertions have a different left‐hand junction with the coding region of rDNA. Each intervening sequence is flanked by a short direct repeat of variable length, being only once present in the uninterrupted rDNA. The intervening sequences of A. lumbricoides show striking similarity to the organization of type I insertion family in dipteran flies, even though they are inserted at different positions in the 26S coding region. Additional rDNA intervening sequences may be present outside of the rDNA cluster, but in not more than 15‐20 homologous copies per haploid genome.

The intervening sequences in the large ribosomal RNA gene of Ascaris lumbricoides var. suum show many similarities to the type I insertions, previously found only in some insect species. They include structural features, but also a presumed transcriptional inactivity in vivo: No transcript of the rDNA intervening sequence in A. lumbricoides could be detected in Northern and dot blot hybridizations. However, the primary structure of the Pol I promoter region is well conserved in interrupted and uninterrupted genes. Moreover, genes with an intervening sequence are correctly initiated in a whole-cell in vitro extract from Ascaris oogonia. Hence, the presence of the intervening sequence alone does not seem to account for a transcriptional inhibition in rRNA genes. As with the type I insertions of insect rDNA, some copies of the A. lumbricoides intervening sequence are also present in locations outside the rDNA cluster. About 50% of the extraribosomal copies are found in a repetitive sequence of the genome, and additional copies are inserted in unique sequences. These striking analogies to type I insertions are discussed, and lead to the conclusion that the two phenomena are undoubtedly related. This is the first report proving the presence of a type I-like insertion element outside of the class Insecta.

An accurate in vitro transcription system which utilizes the cloned 8.8 and 8.4 kb size classes of Ascaris rRNA genes (pAlr8 and pAlr13) and two kinds of cellular extracts from Ascaris oogonia has been established. Both rDNA containing plasmids are efficiently transcribed in vitro by RNA polymerase I from a unique site of rDNA which corresponds to the in vivo initiation site. The in vitro transcription product has a triphosphorylated 5'-end and starts on a G localized 414 bp (pAlr8) upstream of the beginning of the mature 18S rRNA. The promoter region has been delimited by testing the in vitro template activity of a series of restriction fragments. The region essential for the accuracy of initiation is contained within nucleotides -72 to +65, but full efficiency of transcription requires the additional presence of the region from nucleotides +66 to +84. The sequences upstream from position -72 do not appear to modulate the efficiency of specific in vitro initiation. Furthermore, the sequences flanking the transcription initiation site from position -1500 to +570 have been determined in the two cloned representatives of the two rDNA main size classes.

We studied termination of transcription of the ribosomal RNA genes in Ascaris lumbricoides, the first representative in the phylum of nemathelminthes analysed so far. RNase protection experiments in vivo reveal that the 3′ end of the precursor rRNA coincides with the end of mature 26S rRNA. Promoter‐containing miniplasmids are able to direct unique 3′ end formation in vitro at a site identical to that observed in vivo, whereas deletion of these sequences abolishes 3′ end formation throughout the entire spacer. A nuclear run‐on experiment in vitro confirms the drop of polymerase I concentration down‐stream of this site. The termination site for polymerase I transcription of the rDNA operon in A. lumbricoides is therefore unique, and located at the very end of the 26S rRNA gene.

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