doi:10.1016/S0166-6851(02)00219-0 
Copyright © 2002 Elsevier Science B.V. All rights reserved.
Abundant larval transcript-1 and -2 genes from Brugia malayi: diversity of genomic environments but conservation of 5′ promoter sequences functional in Caenorhabditis elegans*1
Natalia Gomez-Escobara, William F. Gregorya, Collette Brittonb, Linda Murrayb, Craig Cortonc, Neil Hallc, Jen Dauba, Mark L. Blaxtera and Rick M Maizels
, 
, a
a Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK
b Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow, UK
c Pathogen Sequencing Unit, Wellcome Trust Sanger Institute Genome Campus, Hinxton, Cambridge, UK
Received 15 July 2002;
revised 2 September 2002;
accepted 2 September 2002. ;
Available online 3 October 2002.
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Abstract
The genomic organisation of two abundant larval transcript (alt) genes from the filarial nematode Brugia malayi has been defined. The products of these genes are 78% identical in amino acid sequence, and are highly expressed in a stage-specific manner by mosquito-borne infective larvae. alt-1 is present as two near-identical copies organised in an inverted repeat of 
7.6 kb, occupying a total of 16 kb of the genome. alt-2 is a single-copy gene at a different locus to alt-1. The two alt-1 genes (alt-1.1 and -1.2) are 99.7% identical in coding sequence and 99.5% in intronic sequences. Both alt-1 and -2 contain 3 introns, and the third intron of alt-2 exhibits a size polymorphism evident in different individual parasites from the laboratory-maintained strain. Genomic sequence up- and down-stream from alt-1.1/1.2 (26 and 6 kb, respectively) and alt-2 (6 and 4 kb, respectively) show that neither gene is in a multiple array or an operon. Most notably, the neighbouring genes of alt-1 and -2 show no similarity to each other, or to the genes flanking the distant alt homologue in Caenorhabditis elegans. Despite this diversity in flanking genes, the 5′ UTR tracts extending some 800 bp upstream of each B. malayi alt gene show a high degree of similarity (overall 59% identity with tracts of 77–86% identity). Surmising that this region may contain conserved promoter elements, constructs containing the B. malayi alt 5′ UTR with or without coding sequence were made fused to β-galactosidase reporter protein. These constructs were injected into the syncytical gonad of C. elegans and progeny stained for β-gal expression. Our results show relatively strong expression in the gut cells of C. elegans for both alt-1 and -2 constructs, commencing in larval worms and continuing into adulthood. Moreover, expression was enhanced when constructs contained segments of alt-1 coding and intronic sequence in addition to the 5′ UTR. We conclude that the high level of alt transcription in filarial L3s is not due to expression from a multi-copy gene family but to a set of strong promoter elements shared between the two alt genes.
Author Keywords: Filarial nematode; Promoter; Transfection; Vaccine antigen
Fig. 1. Polymorphism of the alt-2 gene in B.malayi. DNA samples from 9 individual adult male worms were used as the template with alt-2-specific primers in PCR. The 2 major bands correspond to known variants in the length of intron 3 [10].
Fig. 2. Genomic map of alt-2 based on a lambda bacteriophage clone. Larger boxes represent genes from initiating to terminating codons (including intronic sequences); smaller numbered boxes denote individual predicted exons. Arrows show direction of transcription, and intergenic intervals are indicated. Positions of ESTs from the Filarial Genome Project are indicated, and homologies with C. elegans genes provided. Note that neither of the flanking genes are completely encoded within the phage clone. Gene names are: kat, 3-keto-acyl-CoA thiolase; upt, UP from alt-Two; and nfa, Nuclear Factor IA. The sequence of this genomic clone has been deposited with the Accession Number AY141874.
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Fig. 3. Genomic map of the 50 kb segment containing alt-1.1 and -1.2. BMBAC07G12 comprises alt-1.1, 5 additional predicted genes, and two ribosomal RNA repeat units. BMBAC47K06 overlaps by 5.8 kb, includes the 3′ end of alt-1.1 and the whole of alt-1.2. Solid lines represent sequenced portions of BACs, hatched lines unsequenced. Larger boxes represent genes from initiating to terminating codons (including intronic sequences); smaller numbered boxes denote individual predicted exons. The flecked boxes indicate the positions of the inverted repeat which includes the duplicated alt-1 genes. The numbering commences as indicated, immediately 3′ of a 50-bp repeat segment which could not be unambiguously sequenced. Arrows show direction of transcription, and intergenic intervals are indicated. Gene names are: upo, UP from alt-One; prl, Pleiotropic ReguLator; mog, Masculinization Of the Germ line; alt, Abundant Larval Transcript. Names of C. elegans homologues and their loci in the genome are included. ESTs from the Filarial Genome Project are indicated where appropriate. The accession numbers for each sequence segment are as follows: BMBAC07G12 1–32442, AJ508355; BMBAC47K06, 34494-41950, AF540001; BMBAC47K06 T7 end, 1–2321, AY141875; BMBAC368C11, T7 end, 1–641, BH769870.
Fig. 4. Long-range PCR of B. malayi genomic DNA. Positive amplification was observed when single primers corresponding to the sense strand for alt-1 were used (F, forward), but not when primers corresponded to the antisense strand (R, reverse). Three sets of primers were designed to nt 51–78 (in exon 1), 417–444 (in exon 2), and 909–937 (in intron 3). Lanes on either side marked M contain 1-kb DNA markers. The major products observed were 10, 9 and 8 kb, respectively. Additional products about 3 kb smaller were also observed, which may be due to minor variation in genomic sequence in the nonclonal parasite population used to derive DNA.
Fig. 5. Gene structures for alt-1.1 and -2. Exons are in larger boxes, connected by introns shown as thin lines. Potential 5′ and 3′ regulatory regions are shown as thinner boxes. Motifs showing higher levels of similarity are lettered on dark boxes. The sequences of the 5′ motifs A–E are shown in Fig. 6. Bold numbering is relative to the ATG of each gene; light italic numbering is that of the genomic clones deposited in GenBank™ (Accession numbers given in legends to Fig. 2 and Fig. 3).
Fig. 6. Alignment of the 5′ noncoding regions of alt-1 and -2. The dark boxes represent the 5 motifs with highest similarity between the two genes, notably focussed between 330 and 600 nt upstream from the ATG initiation codons. The open boxes signify promoter motifs which exceeded the criterion for matrix similarity of 0.9 for both alt-1 and -2; the individual scores for each gene are given above and below the sequence respectively, together with the relevant transcription factor. The site of trans-splicing for mature mRNA formation is indicated close to the ATG.
Fig. 7. Genomic environment of the C. elegans alt homologue on cosmids C30G4 and C08A9. Numbering represents Chromosome V positions as retrieved from wormbase (http://www.wormbase.org/). Arrows show direction of transcription, and intergenic intervals are indicated. Note that C. elegans alt does not have a gene number (unlike C08A9.2 for reverse transcriptase) as it was not identified by GeneFinder in the original annotation.
Fig. 8. Promoter analysis in C. elegans. (A) Schematic of 3 constructs in pPD96.04; (B) alt-1 transcriptional construct (NG-033) in L1; (C) alt-1 transcriptional construct (NG-033) in L4; (D) alt-1 transcriptional construct (NG-033) in young Adults; (E) alt-1 translational construct (NG-034) in L2; (F) alt-1 translational construct (NG-034) in Adults; (G) alt-1 translational construct (NG-034) under higher power; (H) alt-2 transcriptional construct (NG-035) in L1.
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