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The hibernation-specific HP-25 gene is expressed specifically in the liver of the chipmunk, a hibernating species of the squirrel family, and exists as a pseudogene in the tree squirrel, a nonhibernating species. Our previous studies have revealed two positively acting transcriptional regulatory regions in the 5'-flanking region of the chipmunk HP-25 gene, one from -260 to -80 and another from -80 to -59, and a pivotal role for hepatocyte nuclear factor-4 (HNF-4), which binds to the proximal regulatory region, in HP-25's liver-specific transcription. A database search for transcription factor binding sites in the distal regulatory region indicated the presence of two potential binding sites for upstream stimulatory factor (USF): one between -161 and -156 and the other between -143 and -138. In an electrophoretic mobility shift assay (EMSA), in vitro-translated USF bound only to the sequence from -143 to -138. USF did not bind the corresponding sequence of the tree squirrel HP-25 gene, which has two base substitutions. Transient transfection studies in COS-7 cells showed that USF could activate the transcription of the chipmunk HP-25 gene, and that tree squirrel-type base substitutions in the USF-binding site aborted the transactivation by USF. By chromatin immunoprecipitation (ChIP) analysis, we confirmed that USF bound to the promoter region of the HP-25 gene in the chipmunk liver, and not in the kidney or heart. These results indicate that USF is involved in the transcriptional regulation of the chipmunk HP-25 gene in the liver, and that the base substitutions in the USF-binding site contribute to the lack of HP-25 gene expression in the tree squirrel.

The chipmunk hibernation-specific protein HP-55 is a component of a 140-kDa complex whose levels are drastically decreased in the blood during hibernation. It is highly homologous to alpha(1)-antitrypsin (AT). In the chipmunk, several alpha(1)-AT-like genes in addition to HP-55 (or CM55-ML) are expressed in the liver and have distinct patterns of regulation during hibernation: in hibernating chipmunks, the level of CM55-ML gene expression is greatly reduced, that of the CM55-MS gene is slightly increased, and the expression of the CM55-MM gene is hardly affected. As a first step towards understanding the hibernation-associated gene regulation of these chipmunk alpha(1)-AT-like genes, we isolated genomic clones for the CM55-ML, CM55-MM, and CM55-MS genes, and analyzed their promoter activities. These alpha(1)-AT-like genes are composed of five exons, and show a similar gene structure to that of the human alpha(1)-AT gene, suggesting that they were generated by the duplication of an ancestral alpha(1)-AT gene. Transient transfection studies using HepG2 and COS-7 cells revealed that for all three alpha(1)-AT-like genes, approximately 150-bp 5' flanking sequences were sufficient for the liver-specific promoter activity, and that the binding of HNF-1 to the promoter region could transactivate transcription. In addition, analysis of the activity of chimeric promoters composed of CM55-ML and CM55-MS gene sequences indicated that the lack of a TATA box-like sequence in the CM55-MS gene is responsible for its weak promoter activity.

The chipmunk hibernation-specific protein HP-27 is a component of the 140-kDa complex that decreases in the blood during hibernation. Although the HP-27 gene is detected in both the chipmunk, a hibernating species of the squirrel family, and the tree squirrel, a nonhibernating species, it is expressed only in the chipmunk, in a liver-specific manner. To understand the difference in HP-27 gene expression between the chipmunk and tree squirrel, we isolated chipmunk and tree squirrel HP-27 genomic clones, and compared their promoter activities. Transient transfection studies in HepG2 cells revealed that the 170 bp 5'-flanking sequence of the chipmunk HP-27 gene was sufficient for liver-specific promoter activity and that deletion of the sequence from -170 to -140 reduced the promoter activity by 90%. Although the corresponding 170 bp 5'-flanking sequence of the tree squirrel HP-27 gene had 89% nucleotide sequence homology to that of the chipmunk, it showed almost no promoter activity in HepG2 cells. In a gel retardation assay using HepG2 or chipmunk liver nuclear extracts, the 5'-flanking sequence of the chipmunk HP-27 gene from -170 to -140 showed a shifted band, but the corresponding tree squirrel sequence did not. Taken together, these data indicate that a transcription factor that binds to this 5'-flanking sequence of the chipmunk HP-27 gene plays an important role in HP-27 gene expression, and the failure of this factor to bind in the case of the tree squirrel HP-27 gene could be responsible for this animal's lack of HP-27 gene expression.

In the chipmunk, a mammalian hibernator, a 140 kDa protein complex found in the blood, drastically decreases in concentration during hibernation. This complex contains four species of proteins, HP-20, -25, -27 and -55. In the present study, cDNA clones coding for the chipmunk HP-55 were isolated from a liver cDNA library. Sequence analysis revealed that HP-55 is produced as a precursor protein of 413 amino acids (aa), that it has a signal peptide of 24 aa, and that it contains four potential N-glycosylation sites. The deduced aa sequence shows 63% identity with that of rat alpha1-antitrypsin (alpha1-AT); however, the sequence corresponding to the reactive center P1-P1' residues was found to be Met-Leu, whereas it is Met-Ser in the rat alpha1-AT. During screening of the chipmunk liver cDNA library, four other related classes of cDNA clones were obtained, each also coding for an alpha1-AT-like protein. In spite of more than 86% overall aa sequence identity among the five chipmunk alpha1-AT-like proteins, they are highly divergent in the putative reactive center region; the putative P1-P1' sequences are Met-Leu (HP-55 or CM55-ML), Met-Met (CM55-MM), Met-Ser (CM55-MS), Ser-Ile (CM55-SI) and Ser-Thr (CM55-ST). Each of the alpha1-AT-like protein mRNAs was expressed in chipmunk liver, and the HP-55 mRNA level was greatly reduced during hibernation. Genomic Southern blot analysis and screening of a liver cDNA library from another hibernating squirrel species, the ground squirrel, also revealed expression of multiple members of the alpha1-AT gene family, whereas analysis of a cDNA library from a non-hibernating species, the tree squirrel, found only a single alpha1-AT gene.

In the acorn barnacle Megabalanus rosa, two types of galactose-binding C-type lectins (BRA-2 and BRA-3) have been identified. Here, we report the isolation and characterization of BRA-2 cDNA and genomic clones. In contrast to the BRA-3 gene, which consists of four exons, BRA-2 is encoded by a single exon, implying differences in the physiological roles of the two lectins.

We have isolated cDNA clones coding for a lectin (BRA-3) from the acorn barnacle, Megabalanus rosa. Sequence comparison of the cDNA clones has revealed polymorphism in the BRA-3 mRNA, which results from single-nucleotide (nt) differences at three positions. All three differences are within the coding region and cause conservative amino acid (aa) changes. The BRA-3 gene is composed of four exons, and the three single-nt differences are located on different exons. In addition, the BRA-3 mRNA and BRA-3 protein levels increased during early summer in a similar fashion, indicating that BRA-3 production is regulated mainly at the level of transcription.

We have purified a rat liver enzyme that catalyzes the NADPH-dependent reduction of 3-deoxyglucosone (3-DG), a major intermediate in the Maillard reaction and a potent cross-linker responsible for the polymerization of proteins. Comparison of the amino acid (aa) sequences of nine peptides obtained from the rat 3-DG-reducing enzyme by lysylendopeptidase digestion with the aa sequence of human aldehyde reductase (ALR) [Bohren et al., J. Biol. Chem. 266 (1991) 24031-24037] strongly suggested that the purified enzyme was rat ALR. We cloned the cDNA encoding ALR from a rat kidney cDNA library using a human ALR cDNA fragment, amplified by polymerase chain reaction, as a probe. All nine peptides identified in the purified rat 3-DG-reducing enzyme were found in the aa sequence deduced from the rat ALR cDNA. Moreover, cell extract from COS-1 cells transfected with the rat ALR cDNA exhibited NADPH-dependent 3-DG-reducing activity and cross-reacted with antiserum raised against the purified rat 3-DG-reducing enzyme. All the above data indicate clearly that the 3-DG-reducing enzyme is identical with ALR. Northern blot analysis of total mRNA from a variety of rat tissues showed fairly high levels of expression of ALR mRNA. This suggests that sufficient ALR is present to detoxify 3-DG when it is formed through the Maillard reaction in vivo.

The number of retrotransposon copia per genome in Drosophila melanogaster cultured cells is two to three times higher than that in D. melanogaster embryo cells. Here, we have found that the genome of the related species, Drosophila simulans, contains in cultured cells more efficiently amplified copia DNA (approximately ten fold). Furthermore, we analyzed copia virus-like particles (VLPs) prepared from D. melanogaster and D. simulans cultured cells, which contain copia RNA and reverse transcriptase (RT) activity, and thus, play a major role in copia replication. The RT activity associated with the D. simulans VLPs was 25 times higher than that associated with the D. melanogaster VLPs. Taken together with the fact that copia is believed to transpose through an RNA intermediate, these results suggest that the amplification of copia DNA should relate to copia RNA-mediated transposition, and the higher RT activity associated with the D. simulans VLPs would lead to the efficient amplification of copia DNA. In a comparison between D. melanogaster and D. simulans copia nucleotide (nt) sequences, five nt substitutions, which cause the respective amino acid changes, were found in the copia RT-coding region. Polymerase chain reaction direct sequencing showed that these five substitutions are the vast majority in each Drosophila species. The substitutions, therefore, may be responsible for the high level of the RT activity associated with the D. simulans VLPs.

The Drosophila melanogaster transposable element copia is usually 5 kb long with long terminal repeats (LTRs), and its major transcripts are a full-length 5-kb RNA and a 2-kb RNA. We have previously shown that the 2-kb RNA is generated through splicing. Here, we have cloned a genomic intronless copia using an oligodeoxyribonucleotide probe which is specific for the junction of the two exons. The unusual copia is bounded by two LTRs and lacks precisely the intron of the 2-kb copia RNA. Identification of genomic intronless copia strongly suggests that copia transposes through an RNA intermediate. Moreover, we have found that copia virus-like particles (VLPs), in which reverse transcription of copia RNA seems likely to occur, packages the spliced copia RNA much less efficiently than the full-length copia RNA. This result leads to the suggestion that much lower copy number of genomic intronless copia, as compared with that of 'normal' copia, may be responsible for the inefficient packaging of the spliced copia RNA into the VLP.

The 5' region of the Japanese encephalitis virus (JEV) RNA was cloned and 3000 nucleotides (nt) were determined by sequencing DNA complementary to viral RNA, and genomic RNA, using oligodeoxynucleotide primers and the dideoxy chain-termination reaction. Comparison of the nt sequence and the reduced amino-acid sequence of JEV with those of other flaviviruses showed significant homologies, which allowed locations to be assigned for three structural proteins.

A DNA fragment which contains the 5'-flanking region of the Saccharomyces cerevisiae enolase 1 gene (ENO1) and a portion of the coding sequence was cloned in a plasmid pMC1587. This fragment was fused in frame to the lacZ gene of Escherichia coli. Many mutants which deleted a portion of the 5'-flanking region of ENO1 were isolated from this ENO1-lacZ fusion plasmid by in vitro recombination. Analysis of beta-galactosidase activity of these mutants indicated that the regulatory region responsible for an efficient expression of the ENO1-lacZ fused gene resides within an 86-bp sequence located at -487 to -402 upstream from the start codon of ENO1. We found that the segment encompassing the 86-bp region worked equally well in an inverted orientation, but the tandem duplication of the sequence did not enhance the expression of the fused gene.