Analysis of mouse Evx genes: Evx-1 displays graded expression in the primitive streak.

Two mouse genes, Evx-1 and Evx-2, each encoding a homeodomain closely related to that of the Drosophila even-skipped gene were isolated using a PCR-based strategy. The structure and sequence of these genes are described. Mapping studies localized Evx-1 to chromosome 6, near the Hox-1 gene cluster, and Evx-2 to chromosome 2, near the Hox-4 cluster. The evolutionary implications of these linkages are discussed. RNA in situ hybridization analysis of Evx expression in embryos demonstrated a striking pattern of Evx-1 expression during gastrulation, whereas Evx-2 RNA could not be detected at any stage by this technique. Evx-1 RNA is first detected shortly before the onset of gastrulation in a region of ectoderm containing cells that will soon be found in the primitive streak. This localized expression of Evx-1 provides the first molecular evidence for regional differences in the mouse embryonic ectoderm before gastrulation. Throughout gastrulation, Evx-1 expression is limited to cells near and within the streak and that expression is graded, with a posterior-to-anterior decrease in the level of RNA. Based on fate-mapping studies indicating that different types of mesoderm emerge from different regions of the primitive streak and our observation that high levels of expression are localized to the region that will give rise to extraembryonic and ventral mesoderm, we speculate that Evx-1 plays a role in the dorsoventral specification of mesodermal cell fate.

Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.

We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.

Identification of DNA sequences required for mouse APRT gene expression.

The mouse aprt promoter contains four GC boxes, which bind transcription factor Spl in vitro, and lacks both TATA and CCAAT boxes. Removal of the two most distal GC boxes of this promoter had little effect on APRT enzyme levels produced in a transient expression assay. Deletion of the distal three GC boxes resulted in a 50% reduction, and deletion of all GC boxes resulted in essentially complete loss of APRT activity. There are two predominant transcription start sites which are located within the region containing the GC boxes. The promoter behaved as a relatively strong promoter when compared to the RSV LTR promoter in a transient CAT assay, and operated in one orientation only. No upstream anti-sense transcripts were detected in either mouse CAK or liver cells, confirming that the mouse aprt promoter, unlike some other GC-rich promoters appears not to support bidirectional transcription.

Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonrandom CpG dinucleotide arrangement.

The functional human adenine phosphoribosyltransferase (APRT) gene is less than 2.6 kilobases in length and contains five exons. The amino acid sequences of APRTs have been highly conserved throughout evolution. The human enzyme is 82%, 90%, and 40% identical to the mouse, hamster, and Escherichia coli enzymes, respectively. The promoter region of the human APRT gene, like that of several other "housekeeping" genes, lacks "TATA" and "CCAAT" boxes but contains five GC boxes that are potential binding sites for the Sp1 transcription factor. The distal three, however, are dispensable for gene expression. Comparison between human and mouse APRT gene nucleotide sequences reveals a high degree of homology within protein coding regions but an absence of significant homology in 5' flanking, 3' untranslated, and intron sequences, except for similarly positioned GC boxes in the promoter region and a 26-base-pair region in intron 3. This 26-base-pair sequence is 92% identical with a similarly positioned sequence in the mouse gene and is also found in intron 3 of the hamster gene, suggesting that its retention may be a consequence of stringent selection. The positions of all introns have been precisely retained in the human and both rodent genes, as has an unusual AG/GC donor splice site in intron 2. Particularly striking is the distribution of CpG dinucleotides within human and rodent APRT genes. Although the nucleotide sequences of intron 1 and the 5' flanking regions of human and mouse APRT genes have no substantial homology, they have a frequency of CpG dinucleotides that is much higher than expected and nonrandom considering the G + C content of the gene. Retention of an elevated CpG dinucleotide content, despite loss of sequence homology, suggests that there may be selection for CpG dinucleotides in these regions and that their maintenance may be important for APRT gene function.

An unusual adenine phosphoribosyltransferase pseudogene is syntenic with its functional gene and is flanked by highly polymorphic DNAs.

A mouse adenine phosphoribosyltransferase (aprt) pseudogene that had previously been recovered from a BALB/c sperm DNA library possessed several unusual features. Its nucleotide sequence, like that of other processed pseudogenes, was colinear with its corresponding mRNA, but it was truncated at its 3' end and lacked a poly(A) tail. The pseudogene was 82% homologous with corresponding regions of the functional gene and had incurred mutations that included transitions, transversions, deletions, and a point insertion. Even though the pseudogene was truncated within the protein-coding region of the corresponding functional gene, it was flanked at both ends by 13-base-pair direct repeats. Curiously, the direct repeats exhibited homology to APRT mRNA at the site of pseudogene divergence. The pseudogene appeared to be common to BALB/c and A/J mice, but it was contained on a 3-kilobase EcoRI fragment in the former strain and a 4.5-kilobase EcoRI fragment in the latter. The BALB/c and apparently the A/J pseudogene both mapped to chromosome 8, which also contains the functional aprt gene. The DNA sequences immediately surrounding the pseudogene in the two strains appeared to be similar, suggesting that the BALB/c and A/J pseudogenes are allelic. However, DNA sequences more distal to the pseudogene in the two strains appeared to vary. Thus, the EcoRI polymorphism was not due to simple loss of an EcoRI site, but was more complex. The pattern of flanking restriction sites was different for each of several enzymes, consistent with extensive DNA rearrangement. Double digests of BALB/c and A/J genomic DNAs revealed complex polymorphisms on both sides of the pseudogene. The results were consistent with insertion, deletion, or other rearrangement of DNA sequences that flank the pseudogene and suggest that this region of mouse chromosome 8 may be a region active for mutation or recombination.

Nucleotide sequence and organization of the mouse adenine phosphoribosyltransferase gene: presence of a coding region common to animal and bacterial phosphoribosyltransferases that has a variable intron/exon arrangement.

We have determined the nucleotide sequence of a functional mouse adenine phosphoribosyltransferase (APRT) gene and its cDNA. The amino acid sequence of the enzyme is deduced from an open reading frame in the cDNA and predicts a protein with a molecular weight of 19,560. The protein coding region of the gene is approximately 2 kilobases, and it is composed of five exons and four introns. While the body of the gene is 53% G + C, the 200 nucleotides upstream from the ATG translation start codon are 66% G + C and contain three copies of the sequence C-C-G-C-C-C. The mouse APRT enzyme shares a homologous 20-amino acid sequence with mouse, hamster, and human hypoxanthine phosphoribosyltransferases (HPRTs) and several bacterial phosphoribosyltransferases. This sequence has previously been shown to be a likely catalytic domain in human HPRT and Escherichia coli glutamine phosphoribosyltransferase. Because of the similarities in function of these proteins, both eukaryotic and prokaryotic, it is not unexpected that they should exhibit one or more regions of homology, particularly at the 5-phosphoribosyl-1-pyrophosphate and purine binding sites, especially if they are related via a common evolutionary lineage. This homologous sequence is interrupted by a single intron in the mouse APRT gene and by two introns in the mouse HPRT gene. Furthermore, the positions of both introns in the HPRT sequence are different from that of the single intron in the corresponding sequence of the APRT gene.

Cloning of a functional human adenine phosphoribosyltransferase (APRT) gene: identification of a restriction fragment length polymorphism and preliminary analysis of DNAs from APRT-deficient families and cell mutants.

A complete human APRT gene has been isolated from a lambda phage genomic library using cloned mouse APRT DNA as a probe. The human gene, contained in a recombinant lambda phage designated lambda Huap15, is functional by virtue of its capacity to transfer human APRT activity to Aprt- mouse recipient cells after phage-mediated transfection. Digestion of lambda Huap15 DNA with BamH1 generated a 2.2-kb fragment that is the only fragment of eight produced to hybridize with the mouse APRT gene. This 2.2-kb BamH1 fragment is a unique, single copy sequence, and has been used to identify a restriction fragment length polymorphism (RFLP) associated with the APRT locus. Taq1 digestion and Southern blot analysis of DNAs from 49 unrelated individuals produced three different patterns. DNAs of 30 individuals produced a restriction pattern of three labeled fragments about 500 bp, 600 bp, and 2.1 kb in size, which is characteristic for individuals homozygous for the more common allele. Two individuals homozygous for the less frequent allele displayed labeled fragments of 500 bp and 2.7 kb. The remaining 17 DNA samples produced all four labeled bands as expected for heterozygous individuals. The frequency of heterozygotes in the population is about 35%, while the frequency of the less common allele is about 0.21. Restriction enzyme analysis of DNAs from two APRT-deficient brothers and from an unrelated heterozygote revealed no gross deletions or rearrangements, nor the Taq1 polymorphism.