Structures of two HaeIII-type genes in the human salivary proline-rich protein multigene family

Two members of the human salivary proline-rich protein (PRP) multigene family have been isolated and completely sequenced. These PRP genes, PRH1 and PRH2, are of the HaeIII-type subfamily and code for acidic PRP proteins. Both genes are approximately 3.5 kilobase pairs (kb) in length and contain four exons. Exon 3 encodes the proline-rich part of the protein and includes five 63-base pair (bp) repeats. CAT and ATA boxes and several possible enhancer sequences occur in a 1-kb region 5' to exon 1. Two sets of repeats occur in the sequenced region in addition to the 63-bp repeats: one pair of about 140 bp flanks 500 bp of DNA in the first intervening sequence, and the other pair of 72 bp is tandemly repeated 1.4 kb 5' to the PRH1 gene. The 4-kb region of sequenced DNA from PRH1 differs by an average of 8.7% from the same region in PRH2, but the nucleotide sequences of the exon 3 of the two genes differ by only 0.2%. This result suggests the occurrence of a recent gene conversion event. The regions containing the 5-fold repeated sequences of 63 bp are identical in the two genes, PRH1 and PRH2. A comparison of the human HaeIII and BstNI subfamily repeats and a comparison of the human, mouse, and rat repeats suggest that the individual repeats have evolved in a concerted fashion within each gene and within the PRP gene family as a whole.     

Structure, organization, and regulation of a hamster proline-rich protein gene. A multigene family

Genomic DNA fragments bearing proline-rich protein (PRP) genes expressed specifically in hamster parotid glands have been isolated and characterized. Complete exonic sequences as well as intronic and a considerable portion of the flanking sequences are reported for a PRP gene, H29. H29 is interrupted by three intervening sequences, with consensus splice junctions, and it likely encodes the acidic hamster PRP Hp43a. Exceedingly high homology of the 5'-untranslated region and the sequence encoding the signal peptide is observed with other PRPs of all species studied. Significant homology was also detected among the repetitive sequences of the mature acidic PRPs from human, mouse, hamster, and rat. This conservation of the internal repeats of the PRPs suggested that proline-rich protein gene evolution involved intragenic duplication of internal repeats and gene duplication and conversion. Both hamster and mouse PRP genes (H29 and mouse proline-rich protein gene, respectively) share considerable sequence similarity in the 5'-flanking regions for about 100 base pairs upstream. The remainder of the upstream sequences were heterologous except for three oligonucleotide regions with 60-70% sequence conservation. These three regions are thought to be involved in the regulation of the tissue-specific PRP gene induction.     

The structure of the gene encoding chain c of the hemoglobin of the earthworm, Lumbricus terrestris

The complete nucleotide sequence of the gene for chain c of hemoglobin of the earthworm Lumbricus terrestris has been determined. The sequence of 4037 base pairs (bp) includes about 310 bp of 5'-flanking sequence and 110 bp 3' to the poly(A) site. Comparison of cDNA and genomic sequences shows four silent differences in codons that suggest the presence of at least two genes. The coding sequence is split by two introns of 1344 and 1169 bp at highly conserved positions (Jhiang, S. M., Garey, J. R., and Riggs, A. F. (1988) Science 240, 334-336). The first intron possesses the unusual 5' splice junction sequence GC instead of GT. Many tandem triplet repeats based on (GAT) and (CCT) are present in the first intron. The second intron has nine tandem repeats based on the consensus sequence AAGGAAGGAGGTC. Each intron has several exact inverted repeats of 9-10 bp that might result in loops of 78-140 nucleotides in the RNA prior to splicing. The sequences in the second intron, at positions 2423-2644 are about 65% identical with parts of several genes found in yeast mitochondria and in DNA from several other organisms.     

PCR Amplification and Sequencing of a Wheat rbc S Gene Promoter

The promoter region of the rbc S gene from Triticum aestivum L. FAN-61 was amplified by PCR. Two commonly used restriction enzyme sites, Barn HI and Sma Ⅰ, were respectively added to the 5′ and 3′ ends of the promoter fragment in order to make it easier for subsequent manipulation. The amplified fragment was cloned into pUC18/19 vectors and its sequence was determined. The 704 bp promoter fragment contained a TATA Box (TATATATA) and an Ⅰ Box (GATAAT). The sequence of CCAAC in it was similar to the sequence of CCAAT found upstream of other eukaryotic genes. Moreover, it also contained the consensus sequences of CCACA and GAACGTGAGCCA which were present in those of other monocot rbc S.     

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species.

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.     

Complete nucleotide sequence of a functional HLA-DP beta gene and the region between the DP beta 1 and DP alpha 1 genes: comparison of the 5'' ends of HLA class II genes.

The complete nucleotide sequence of an HLA-DP beta 1 gene and part of the adjacent DP alpha 1 gene, up to and including the signal sequence exon, were determined. The sequence of the DP beta 1 gene identified it as the DPw4 allele. The six exons of the DP beta 1 gene spanned over 11,000 bp of sequence. The arrangement of the gene was broadly analogous to genes of other class II beta chains. The beta 1 exon was flanked by introns of over 4 kb. Comparisons with published sequences of cDNA clones indicated that an alternative splice junction, at the 3' end of the gene, is used in at least one allele. Variation in choice of splice junction indicates an additional mechanism for allelic variation in class II genes. The sequence also indicated that the DP beta 1 and DP alpha 1 genes are separated by only 2 kb at their 5' ends. Comparison of the 5' ends of the DP alpha 1 and beta 1 genes with other class II sequences, including the DZ alpha gene, showed conservation of several blocks of sequences thought to be involved in control of expression. Some areas of the introns were partially conserved in the DQ beta gene, and several other intron sequences were homologous to sequences found in other unrelated genes.     

Isolation and characterization of the three Waxy genes encoding the granule-bound starch synthase in hexaploid wheat.

Complete genomic DNA sequences of three homoeologous Waxy structural genes, located on the chromosomes 7A, 4A, and 7D in hexaploid wheat (Triticum aestivum L. cv. Chinese Spring), were separately determined and analyzed. Those structural genes in lengths from start to stop codon were 2781bp in Wx-7A, 2794bp in Wx-4A, and 2862bp in Wx-7D, each of which consisted of 11 exons and ten introns. They were closely similar to one another in the nucleotide sequences, with 95.6-96.3% homology in mature protein regions, 88. 7-93.0% in transit-peptide regions, and 70.5-75.2% in the introns. These wheat Waxy genes were GC-rich when compared with standard values for plant genomes reported so far. This was reflected in the extremely high G/C occupation frequency at the third position of the codons in the coding regions. The sequence divergence in the exon regions was mostly due to the substitution of nucleotides, whereas that found in the introns was attributed to substitution, insertion and/or deletion of nucleotides. Only the Wx-4A gene contained a trinucleotide insertion (CAA) in the region encoding the transit peptide. Most of the substitutions observed in the exon regions were categorized as synonymous, and higher sequence similarities (96.5-97. 4%) were conserved at the protein level. The phylogenetic tree obtained in terms of the amino acid sequence variations showed a well-resolved phylogenetic relationship among wheat Waxy genes and those from other plants.     

Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.

The Spec1 and Spec2 genes of Strongylocentrotus purpuratus are closely associated with the differentiation of aboral ectoderm. To examine cis-regulatory elements involved in the spatial expression of the Spec genes, we fused the Escherichia coli lacZ gene containing a nuclear targeting signal to 5'flanking DNA plus 5' untranslated leader sequences from Spec1, Spec2a, and Spec2c. All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions. The Spec-lacZ reporter gene plasmids were microinjected into eggs of S. purpuratus, Lytechinus variegatus, and L. pictus, and beta-galactosidase activity was determined in situ by X-gal staining. The Spec2a-lacZ fusion gene, which contained 1516 bp of 5' flanking DNA and 18 bp of 5' untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species. The Spec1-lacZ fusion gene was expressed in a strikingly different fashion--preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells. The staining pattern was the same in either homologous or heterologous embryos. The Spec2c-lacZ fusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed. To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5' flanking DNA from the Spec2a-lacZ fusion gene, resulting in a delta Spec2a-lacZ fusion gene that contained only the conserved DNA region. This gene fusion showed preferential expression in aboral ectoderm cells. However, the cell type specificity was not as great as with the parental Spec2a-lacZ plasmid. These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between -1516 and -697 bp in the 5' flanking DNA of Spec2a.     

Structure and evolution of the human prosaposin chromosomal gene.

The gene for prosaposin was characterized by sequence analysis of chromosomal DNA to gain insight into the evolution of this locus that encodes four highly conserved sphingolipid activator proteins or saposins. The 13 exons ranged in size from 57 to 1200 bp, while the introns were from 91 to 3812 bp in length. The regions encoding saposins A, B, and D each had three exons, while that for saposin C had only two. This sequence included the regions that encode the carboxy terminus of the signal peptide, the four mature prosaposin proteins, and the 3' untranslated region. Primer extension studies indicated that over 99% of the coding sequence was contained in these 19,985 bp. Use of PCR and reverse PCR techniques indicated that the most 5' coding approximately 140 bp contained large introns and at least two small exons. Analyses of the intronic positions in the saposin regions indicated that this gene evolved from an ancestral gene by two duplication events and at least one gene rearrangement involving a double crossover after introns had been inserted into the gene.