The human alpha-fetoprotein gene. Sequence organization and the 5' flanking region

The human alpha-fetoprotein (AFP) gene was isolated into three overlapping clones in bacteriophage lambda vectors and its sequence organization analyzed by restriction endonuclease mapping and nucleotide sequencing. The human AFP gene is about 20 kilobase pairs long and contains 15 exons and 14 introns. The overall organization of the human AFP gene is similar to that of the mouse AFP gene, with all but two exons showing identical sizes. Nucleotide sequences at all exon/intron junctions display similarity to the consensus boundary sequence (Breathnach, R., and Chambon, P. (1981) Annu. Rev. Biochem. 50, 349-383), with the GT-AG rule applied to the splicing point. The cap site maps 44 nucleotides upstream from the translation initiation site. The "TATA box" is located 27 nucleotides upstream from the putative cap site and is flanked by sequences with dyad symmetry. The TATA box can thus be placed in the loop portion of a possible stem-loop structure formed by intrastrand base-pairing. Other characteristic nucleotide sequences in the 5' flanking region include a CCAAC pentamer, a 14-base pair (bp) enhancer-like sequence, and a 9-bp sequence homologous to the glucocorticoid responsive element. A long (90 bp) direct repeat and several alternating purine/pyrimidine sequences are also present in the 5' flanking region. A 736-bp sequence of the 5' flanking region adjacent to the cap site of the human AFP gene shows a 61% similarity with the corresponding region of the mouse AFP gene. There are two Alu family sequences and two poly(dT-dG) repeats in the human AFP gene that show different distribution patterns from those in the mouse AFP gene.     

Structure and expression of mouse apolipoprotein E gene

The mouse apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. DNA including apolipoprotein E gene plus segments 2.5 kilobases upstream and 0.3 kilobase downstream of the coding region was transfected into NIH3T3 cells. The cells expressed the same-size apolipoprotein E mRNA and protein as those produced by mouse endogenously. The nucleotide sequence of the gene plus 5' and 3' flanking regions (one kilobase each) was determined. The sequence of the mouse apoliprotein E gene was highly homologous to that of the rat gene, not only in the coding regions but also in the non-coding and intron regions. The mouse and the human apolipoprotein E genes were homologous in the 5' proximal flanking region up to about 200 nucleotides as well as in the four exons. This proximal region was highly conserved for the genes of mouse, rat and human; the relative positions of the "TATA box" and the two copies of "GC box" were identical.     

Tissue- and developmental stage-specific expression of the rat ornithine carbamoyltransferase gene in transgenic mice

Rat ornithine carbamoyltransferase (OCT; EC 2.1.3.3) is encoded by a large gene of 75 kilobases. Expression of this gene is restricted to the liver and small intestine, and there is an increase in expression late in gestation. The recombinant gene carrying 1.3 kilobases of the 5' flanking region of the gene fused to the rat OCT cDNA was microinjected into fertilized eggs, and 17 transgenic mice were produced. Expression in the liver of the transgene was detected in three mice. In these mice, the transgene expression was observed exclusively in the liver and small intestine. Expression of the transgene in the intestine was comparable to that of the endogenous mouse OCT gene, whereas expression in the liver was much lower than that of the endogenous gene. The developmental pattern of expression of the transgene was similar to that of the endogenous gene. Therefore, the 5' flanking sequence of the rat OCT gene seems to be sufficient for the developmental and tissue-specific expression of the gene. An explanation for low expression in the liver remains the subject of ongoing study.     

Nucleotide sequence of the gene for human factor IX (antihemophilic factor B)

Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.     

Role of alpha-fetoprotein regulatory elements in transcriptional activation in transient heterokaryons.

The requirements for activation of the mouse alpha-fetoprotein (AFP) gene in transient heterokaryons were investigated. For this purpose, the 7-kilobases of DNA flanking the 5' end of the AFP gene were linked to a mouse major histocompatibility complex (MHC) class I structural gene. The fusion gene was stably integrated at different sites into mouse L-cells, which do not transcribe the AFP gene. Transient heterokaryon fusions demonstrated that the silent AFP-MHC gene and the endogenous AFP gene were activated by factors present in HepG2 cells, a liver-derived cell line, but not by those in HeLa cells. Activation was detected at the protein level in single heterokaryons by using monoclonal antibodies against the cell surface protein and at the mRNA level in populations of cells. The AFP promoter alone was sufficient for activation could be used for DNA transfer strategies to identify genes which can activate AFP promoter elements in trans.     

Clusters of genes encoding mouse transplantation antigens

We constructed a cosmid library from BALB/c mouse sperm DNA and isolated 64 cosmid clones with cDNA probes for transplantation antigens (class I molecules). Of these clones, 54 mapped into 13 gene clusters containing 36 distinct class I genes and encompassing 837 kilobases of DNA. One gene cluster mapped to the L region and a second cluster with seven genes to the Qa-2,3 region of the major histocompatibility complex. Restriction map and Southern blot analyses suggest that there are subgroups of class I genes. Using a 5' flanking sequence of the L gene as a hybridization probe, we show the L gene to be present in mouse strains expressing this antigen but deleted or mutated in strains failing to express it. Our data suggest that gene duplication and deletion presumably by homologous but unequal crossing-over has altered the size and organization of the class I clusters in different mouse strains and probably is an important mechanism for generating polymorphism in these genes. Analysis of the 36 class I genes with cDNA probes specific for the 5' and 3' ends shows that the exon encoding the third external domain is far more conserved than those encoding the first and second external domains of the transplantation antigen. These differences in variability have interesting functional implications.     

Chromosomal arrangement of leghemoglobin genes in soybean.

A cluster of four different leghemoglobin (Lb) genes was isolated from AluI-HaeIII and EcoRI genomic libraries of soybean in a set of overlapping clones which together include 45 kilobases (kb) of contiguous DNA. These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-Lb psi-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus. A sequence probably common to the 3' region of Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main Lb locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is primarily expressed in root and leaf is found 5' to two Lb loci. Overall, the main leghemoglobin locus is similar in structure to the mammalian globin gene loci.