Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene

The human apo-E gene has been isolated from a lambda phage library using as a probe the previously reported apo-E cDNA clone pE-301. Lambda apo-E was mapped and subcloned, and the apo-E gene was completely sequenced. The DNA sequence was compared with that of a near full length cDNA clone pE-368 and revealed three introns. The first intron was in the region that corresponds to the 5' untranslated region of apo-E mRNA. The second intron interrupted the codon specifying amino acid -4 of the apo-E signal peptide. The third intron interrupted the codon specifying amino acid 61 of the mature protein. Analysis of the DNA sequence revealed four Alu sequences. Two were in opposite orientations in the second intron, and one each occurred in the regions 5' and 3' to the apo-E gene. There were two base differences between the apo-E gene sequence and the sequence derived from the cDNA clones. At the codon for amino acid residue 112, the apo-E gene contained CGC, specifying Arg, whereas the cDNA contained TGC, specifying Cys. The other base difference was in the area corresponding to the 5' untranslated region of apo-E mRNA. Apo-E is commonly polymorphic in the population and the data suggest that the genomic clone was derived from the epsilon 4 apo-E allele, whereas the cDNA clones were derived from the epsilon 3 apo-E allele. S1 nuclease protection and primer extension experiments allowed the tentative assignment of the cap site of apo-E mRNA to the A approximately 44 base pairs upstream of the GT that begins the first intron. The sequence TATAATT was identified beginning 33 base pairs upstream of the proposed cap site and is presumably one element of the apo-E promoter. Finally, the apo-E gene was mapped in the human genome to chromosome 19 through the use of DNA probes and human-rodent somatic cell hybrids.     

Co-inheritance of specific genotypes of HSPG and ApoE gene increases risk of type 2 diabetic nephropathy

The objective of this paper is to investigate co-inheritance of specific HSPG and ApoE genotypes in the development of Chinese type 2 diabetic nephropathy. PCR-RFLP was used to detect HSPG and ApoE genotypes in 385 Chinese subjects including 298 patients with type 2 diabetes mellitus (T2DM) and 87 non-diabetic controls (Non-DM). The T2DM group was subdivided into patients with (TDN; n = 218) and without diabetic nephropathy (Non-DN; n = 80). The latter group was further subdivided into groups of patients with microalbuminuria nephropathy (DN-1; n = 129) and severe diabetic nephropathy (DN-2; n = 89). We then compared the relative frequencies of various HSPG and ApoE genotypes and alleles among the groups, searching for predictive trends. The T allele of the HSPG gene occurred more frequently in the DN-2 group than in the Non-DN or DN-1 or groups, their (Fisher's exact p was 1.05 × 10^–3 and 6.58 × 10^–6; odds ratios were 2.09 (95% CI 1.32–3.30) and 2.48 (95% CI 1.64–3.74), respectively. The E2 allele of the ApoE gene occurred more frequently in the T2DM than in the Non-DM group, the Fisher's exact p was 0.0087; odds ratio was 3.45 (95% CI 1.30–9.81). Genotype analysis showed that the TT or TG of HSPG gene were paired with the E2/2 or E2/3 of ApoE gene significantly more frequently in the TDN group than in the Non-DN group, with an odds ratio of 3.03 (95% CI 1.03–8.90). There was no significant differences in other combinations of genotypes in HSPG and ApoE genes between TDN and Non-DN group. These results suggest that the HSPG T allele is a risk factor for the development of severe diabetic nephropathy in type 2 diabetic patients, and that the ApoE E2 allele is a risk factor for the occurrence of type 2 diabetes mellitus in Chinese general population. In addition, we find that co-inheritance of T/E2 confers a higher risk of type 2 diabetes mellitus progression to diabetic nephropathy in Chinese.     

Apolipoprotein E polymorphism in normal Han Chinese population: frequency and effect on lipid parameters

Apolipoprotein E (ApoE) genotypes were studied in order to determine the prevalence and effect on lipid parameters in normal Han Chinese population. Fragments of ApoE gene forth exon containing codon 112 and 158 polymorphic locus were amplified by PCR, and then digested with Cfo I endonuclease. Genotypes and alleles frequencies of 168 healthy Han Chinese were calculated. The frequency of genotypes ε3/3, ε3/4, and ε2/3 was found to be 75.00, 10.70, and 11.90%, respectively, and 0.60, 1.20, and 0.60% for ε2/2, ε2/4, and ε4/4. The effects of ApoE genotypes and alleles on lipid parameters were analyzed. The effects of ApoE alleles on TC, LDL-C, ApoB was: along a decreasing gradient ε4 > ε3 > ε2. The effect of ε4 allele was to increase serum levels of TC, LDL-C and ApoB, and ε2 allele had an effect opposite to that of ε4 allele. Results obtained in this study indicate that ApoE polymorphism is an independent genetic factor on individual serum levels of lipids and apolipoproteins. Shu Liang and Min Pan should both be considered first authors.     

Activation of N-ras in a human melanoma cell line.

DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.     

Two coding regions closely linked to the rat apolipoprotein E gene: nucleotide sequences of rat apolipoprotein C-I and ECL cDNA.

Restriction fragments isolated from a 17-kb rat genomic DNA clone containing the gene for apolipoprotein (apo) E were radiolabeled and used to screen a rat liver cDNA library. A cDNA clone hybridizing to a 6-kb genomic DNA fragment was isolated and the nucleotide sequence of the cDNA insert determined. The sequence was homologous to the sequence for human apo C-I and was used to derive the corresponding amino acid sequence. Unlike human apo C-I, mature rat apo C-I contains histidine, lacks valine, and has alanine at the C terminus and aspartate as the N terminus. Screening the rat liver cDNA library with a radiolabeled 1.9-kb restriction fragment from the genomic DNA clone containing the rat apo E gene identified another cDNA clone (ECL cDNA). Nucleotide sequencing yielded a derived 75-amino-acid sequence for the ECL protein with a hydrophobicity profile similar to that of rat apo C-I. Northern analysis demonstrated a 0.50-kb band for ECL mRNA. The tissue-specific expression of the gene is similar to that of rat apo C-I. This study indicates that the rat apo C-I and ECL genes are closely linked, about 4.5 and 12 kb downstream of the apo E gene, respectively.     

An efficient and rapid method for cDNA cloning from difficult templates using codon optimization and SOE-PCR: with human RANK and TIMP2 gene as examples

As gene cloning from difficult templates with regionalized high GC content is a long recognized problem, we have developed a novel and reliable method to clone such genes. Firstly, the high GC content region of the target cDNA was synthesized directly after codon optimization and the remaining cDNA fragment without high GC content was generated by routine RT-PCR. Then the entire redesigned coding sequence of the target gene was obtained by fusing the above available two cDNA fragments with SOE-PCR (splicing by overlapping extension-PCR). We have cloned the human RANK gene (ten exons; CDS 1851 bp) using this strategy. The redesigned cDNA was transfected into an eukaryotic expression system (A459 cells) to verify its expression. RT-PCR and western blotting confirmed this. To validate our method, we also successfully cloned human TIMP2 gene (five exons; CDS 660 bp) also having a regionalized high GC content. Our strategy for combining codon optimization and SOE-PCR to clone difficult genes is thus feasible and potentially universally applicable.     

ApoE −491A/T Promoter Polymorphism is not an Independent Risk Factor,but Associated with the ε4 Allele in Hungarian Alzheimer’s Dementia Population

Apolipoprotein E gene (Apoε) has three common alleles (ε2, ε3, and ε4), of which ε4 has been shown to be associated with an increased risk for Alzheimer’s disease (AD). Possible additional genetic factors, like the −491A variant of ApoE promoter may modify the development of AD, independently of the ApoE allele status. The objective of this study was to investigate whether A/T allelic polymorphism at site−491 of the ApoE promoter is associated with AD in a Hungarian population. The genomic DNA isolated from peripheral blood lymphocytes of 52 late-onset AD and 53 control individuals was used as a template for the two examined polymorphisms and PCR assay was applied. The ε4 allele was significantly over-represented in the AD group (28%) as compared with the control population (7%). No significant differences have been found between the control and the AD populations regarding the occurrence of the promoter A allele frequencies (control: 77%, AD: 70%). However, the AA genotype was more frequent in the AD group (48%) than in the control (10%) when the presence of ε4 allele was also considered. It is unlikely therefore that the −491A variant of the ApoE promoter gene is an independent risk factor in the Hungarian AD population, but a linkage disequilibrium exists between the two examined mutations.     

Apolipoprotein E deficiency promotes increased oxidative stress and compensatory increases in antioxidants in brain tissue

The epsilon 4 allele of the apolipoprotein E gene (ApoE) is associated with Alzheimer's disease (AD). The extent of oxidative damage in AD brains correlates with the presence of the E4 allele of ApoE, suggesting an association between the ApoE4 genotype and oxygen-mediated damage in AD. We tested this hypothesis by subjecting normal and transgenic mice lacking ApoE to oxidative stress by folate deprivation and/or excess dietary iron. Brain tissue of ApoE-deficient mice displayed increased glutathione and antioxidant levels, consistent with attempts to compensate for the lack of ApoE. Folate deprivation and iron challenge individually increased glutathione and antioxidant levels in both normal and ApoE-deficient brain tissue. However, combined treatment with folate deprivation and dietary iron depleted antioxidant capacity and induced oxidative damage in ApoE-deficient brains despite increased glutathione, indicating an inability to compensate for the lack of ApoE under these conditions. These data support the hypothesis that ApoE deficiency is associated with oxidative damage, and demonstrate a combinatorial influence of genetic predisposition, dietary deficiency, and oxidative stress on oxidative damage relevant to AD.     

载脂蛋白多基因家族分子进化的研究

与脂质运输有关的载脂蛋白基因构成一个复杂的多基因家族。为探讨这种演化时间长的基因家族的进化规律,本文首先建立了一种在非均衡进化速率条件下计算系统发生树中任意分支长度的简易方法,并可在此基础上算出无根分支系统树中分歧年代的期望值。进一步对本文科１０个种属共２６种载脂蛋白的系统演作作了实际分析,结果提示：①ＡｐｏＡ－Ｉ＇ＡｐｏＡ－ＩＶ,ＡｐｏＥ及ＡｐｏＡ－ＩＩ的共同祖先可能在奥陶纪水生脊椎动物中就已存     

Human urate oxidase gene: cloning and partial sequence analysis reveal a stop codon within the fifth exon

Using the cDNA and selected genomic probes of rat urate oxidase, we have screened the human genomic library and isolated seven clones; one clone (clone 13) contained exonic regions which correspond to the exons 5, 6, and 7 of rat urate oxidase gene. The nucleotide sequence was determined for these three exons and exon/intron junctions, and compared with the sequence from the rat gene. A mutation resulting in a stop codon TGA was found in the fifth exon of the human urate oxidase gene. Sequence analysis of the polymerase chain reaction amplified DNA, corresponding to the fifth exon of urate oxidase from DNA samples from four different individuals, confirmed the same TGA stop codon in all. This single stop codon mutation and/or other mutation(s) in this gene may be responsible for the lack of urate oxidase activity in the human.     

Use of a gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT.

Pseudomonas aeruginosa can convert to a mucoid colony morphology by a genetic mechanism called alginate conversion; this results in the production of copious amounts of the exopolysaccharide alginate. The mucoid phenotype of P. aeruginosa is commonly associated with its ability to cause chronic pulmonary tract infections in patients with cystic fibrosis. In this study we isolated the cis-acting locus involved in alginate conversion, called algS, from both mucoid and nonmucoid isogenic strains. We then examined the role of algS in the control of algT, a trans-active gene required for alginate production in P. aeruginosa. We used a new cosmid cloning vector, called pEMR2, that permitted both the cloning of large DNA fragments and their subsequent gene replacement in P. aeruginosa. To verify the predicted properties of this vector, we isolated and tested a pEMR2 hisI+ clone. Using cloned algS-containing DNA and a method for gene replacement, we constructed isogenic strains of P. aeruginosa that had Tn501 adjacent to algS on the chromosome. Two pEMR2 clone banks containing genomic fragments from isogenic algS(On) (exhibiting the alginate production phenotype) and algS(Off) (exhibiting the non-alginate production phenotype) strains were constructed, and Tn501 served as an adjacent marker to select for clones containing the respective algS allele. The pEMR2 algS(On) and pEMR2 algS(Off) clones were shown to contain the indicated algS allele by gene replacement with the chromosome of strains that carried the opposite allele. To test whether algS controls the expression of the adjacent algT gene, we constructed a pLAFR1 algS(Off)T clone and showed it to be unable to complement an algT::Tn501 mutation in trans. In contrast, a pLAFR1 algS(On)T clone did complement algT::Tn501 in trans. Thus, algS appears to control the activation of algT expression, bringing about alginate conversion.     

Expression of human thymidylate synthase in Escherichia coli

A cDNA clone encoding thymidylate synthase (TS) has been isolated from a human T-cell library and modified in the 5'-untranslated region to incorporate several unique cloning sites. The gene has been cloned as a cassette into several Escherichia coli expression vectors which did not provide detectable amounts of the enzyme. A successful approach used a constitutive E. coli expression vector developed for the enzyme from Lactobacillus casei. A 115-base pair 5'-untranslated region from the L. casei TS which contains a ribosomal binding site and other regulatory sequences has been fused to the coding region of the human TS gene to provide a construct that is expressed in E. coli. The level of expression was further enhanced by altering the nucleotide sequence of the first 90 base pairs to accommodate common codon use in E. coli. In our best expression system, catalytically active human TS is expressed to a level that represents about 1.6% of the total soluble protein. The recombinant human TS has been purified and characterized; except for the presence of an amino-terminal blocking group, the enzyme has physical and kinetic properties similar to the enzyme isolated from human cells.     

Isoform-Specific Effects of Apolipoprotein E on Markers of Inflammation and Toxicity in Brain Glia and Neuronal Cells In Vitro

Mutations to the cholesterol transport protein apolipoprotein E (ApoE) have been identified as a major risk factor for the development of sporadic or late-onset Alzheimer’s disease (AD), with the e4 allele representing an increased risk and the rare e2 allele having a reduced risk compared to the primary e3 form. The reasons behind the change in risk are not entirely understood, though ApoE4 has been connected to inflammation and toxicity in both the brain and the periphery. The goal of this study was to better understand how the ApoE isoforms (ApoE2/3/4) confer differential AD-related risk by assessing cell-specific ApoE-related neuroinflammatory and neurotoxic effects. We compared the effects of ApoE isoforms in vitro on human astrocytes, a human immortalized microglia cell line (HMC3), and the human neuroblastoma cell line SH-SY5Y. Cells were treated for 24 h with or without recombinant ApoE2, ApoE3, or ApoE4 (20 nM) and inflammation and toxicity markers assessed. Our results indicated the expression of inflammatory cytokines IL-1β, TNFα, and IL-6 in human astrocytes was increased in response to all ApoE isoforms, with ApoE4 evoking the highest level of cytokine expression. In response to ApoE2 or ApoE3, microglial cells showed reduced levels of microglial activation markers TREM2 and Clec7a, while ApoE4 induced increased levels of both markers. ApoE2 promoted neuron survival through increased BDNF release from astrocytes. In addition, ApoE2 promoted, while ApoE4 reduced, neuronal viability. Overall, these results suggest that ApoE4 acts on cells in the brain to promote inflammation and neuronal injury and that the deleterious effects of ApoE4 on these cells may, in part, contribute to its role as a risk factor for AD.     

Apolipoprotein E (ApoE) polymorphism is related to differences in potential fertility in women: a case of antagonistic pleiotropy?

The alleles that are detrimental to health, especially in older age, are thought to persist in populations because they also confer some benefits for individuals (through antagonistic pleiotropy). The ApoE4 allele at the ApoE locus, encoding apolipoprotein E (ApoE), significantly increases risk of poor health, and yet it is present in many populations at relatively high frequencies. Why has it not been replaced by natural selection with the health-beneficial ApoE3 allele? ApoE is a major supplier of cholesterol precursor for the production of ovarian oestrogen and progesterone, thus ApoE has been suggested as the potential candidate gene that may cause variation in reproductive performance. Our results support this hypothesis showing that in 117 regularly menstruating women those with genotypes with at least one ApoE4 allele had significantly higher levels of mean luteal progesterone (144.21 pmol l⁻¹) than women with genotypes without ApoE4 (120.49 pmol l⁻¹), which indicates higher potential fertility. The hormonal profiles were based on daily data for entire menstrual cycles. We suggest that the finding of higher progesterone in women with ApoE4 allele could provide first strong evidence for an evolutionary mechanism of maintaining the ancestral and health-worsening ApoE4 allele in human populations.     

Human apolipoprotein E isoform-specific differences in neuronal sprouting in organotypic hippocampal culture

The apolipoprotein E (ApoE) epsilon4 allele is a major risk factor for neurodegenerative conditions, including Alzheimer's disease. A role for ApoE is implicated in regeneration of synaptic circuitry after neural injury. In the in vitro mouse organotypic hippocampal slice culture system, we previously showed that cultures derived from ApoE-knockout mice are defective in mossy fiber sprouting into the dentate gyrus molecular layer. This sprouting defect was rescued in cultures from transgenic mice expressing ApoE3 under the control of the human promoter and in ApoE-knockout cultures treated with ApoE3-conditioned media. Although the ApoE3 transgene fully restored sprouting, ApoE4 restored sprouting to only 58% of ApoE3 levels. These data indicate that ApoE isoform-specific effects on neuroregeneration may contribute to its genetic risk for Alzheimer's disease.     

Apolipoprotein E Gene Polymorphism and Allele Frequencies in the Lebanese Population

Apolipoprotein E (ApoE) genotypes were studied in order to determine the prevalence in the Lebanese population and compare it with other populations. DNA from 160 unrelated healthy donors from our HLA-bank was used. ApoE genotype was determined using the CardioVascular Disease (CVD) StripAssay (this assay is based on a Polymerase Chain Reaction-Reverse Hybridization technique). The prevalence of genotypes E3/3, E3/4, and E2/3 was found to be 69%, 26%, and 22%, respectively, and 0.6% for each of E2/4 and E4/4 genotypes. The Lebanese population tested showed similarities to earlier reported ApoE genotypic distributions (high E3 allele frequency) but also peculiar differences especially to some Arabic countries (total absence of E2 allele among Saudis) and other populations. This is the first report from Lebanon that will serve as a template for future investigations of the prevalence of ApoE alleles in association with various clinical entities.     

Cloning, nucleotide sequence, and expression of the flavodoxin gene from Desulfovibrio vulgaris (Hildenborough)

The gene coding for the flavodoxin protein from Desulfovibrio vulgaris (Hildenborough) has been identified, cloned, and sequenced. DNA fragments containing the flavodoxin gene were identified by hybridization of a mixed synthetic heptadecanucleotide probe to Southern blots of SalI-digested genomic DNA. The nucleotide sequences of the probe were derived from the published protein primary structure (Dubourdieu, M., LeGall, J., and Fox, J. L. (1973) Biochem. Biophys. Res. Commun. 52, 1418-1425). The same oligonucleotide probe was used to screen libraries (in pUC19) containing size-selected SalI fragments. One recombinant, carrying a 1.6-kilobase (kb) insert which strongly hybridizes to the probe, was found to contain a nucleotide sequence which codes for the first 104 residues of the amino-terminal portion of the flavodoxin protein sequence but lacked the remainder of the gene. Therefore, a PstI restriction fragment from this clone was used as a probe to isolate the entire gene from a partial Sau3AI library in Charon 35. Of the plaques which continued to hybridize strongly to this probe through repeated screenings, one recombinant, containing a 16-kb insert, was further characterized. The entire flavodoxin gene was localized within a 1.4-kb XhoI-SacI fragment of this clone. The complete nucleotide sequence of the structural gene for the flavodoxin protein from Desulfovibrio vulgaris and flanking sequences which may include promoter and regulatory sequences are reported here. The cloned flavodoxin gene was placed behind the hybrid tac promoter for overexpression of the protein in Escherichia coli. Modification to the 5'-end of the gene, including substitutions at the second codon, were required to obtain high levels of expression. The expressed recombinant flavodoxin protein is isolated from E. coli cells as the holoprotein with physical and spectral properties similar to the protein isolated from D. vulgaris. To our knowledge, this is the first example of the expression of a foreign flavodoxin gene in E. coli using recombinant DNA methods.