Complex events in the evolution of the haptoglobin gene cluster in primates

Southern blot analyses of genomic DNA show that new world monkeys have only one haptoglobin gene but that chimpanzees, gorillas, orangutans, and old world monkeys have three. Humans have two: haptoglobin (Hp) and haptoglobin-related (Hpr). These observations suggest that a triplication of the haptoglobin locus occurred after the divergence of the new world monkeys, followed by a deletion of one locus in humans. To investigate these events, we have cloned the haptoglobin gene cluster in chimpanzee. The organization of the Hp and Hpr genes in chimpanzees is the same as in humans, including a retrovirus-like sequence in the first intron of Hpr. The third gene, which we name Hpp for haptoglobin primate, is 16 kilobases downstream of Hpr. A second copy of the retrovirus-like sequence occurs between Hpr and Hpp. The nucleotide sequence of the chimpanzee Hpp gene suggests that it may code for a functional protein, but the chimpanzee Hpr gene has a single base deletion in exon 5 that causes a frameshift. Comparison of the human and chimpanzee sequences suggests that the human Hpr gene was generated by a homologous unequal crossover between ancestral Hpr and Hpp genes. The crossover point lies within a 1.3-kilobase region containing exon 5 and 500 nucleotides 3' to the genes, but the exact point is obscured by a subsequent gene conversion event.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

Nucleotide sequence of the haptoglobin and haptoglobin-related gene pair. The haptoglobin-related gene contains a retrovirus-like element

Thirty-three kilobase pairs (kb) of human DNA containing the haptoglobin (Hp) and haptoglobin-related (Hpr) gene pair were cloned, and the nucleotide sequence of 21-kb DNA was determined. The two genes are closely linked, with Hpr being 2.2 kb downstream of Hp. Six hundred nucleotides of DNA occur between the two genes that are not found either 5' to the Hp gene or 3' to the Hpr gene. After the duplication event, the first intron of the Hpr gene acquired a 9-kb insert consisting mainly of a retrovirus-like element with a potential primer-binding site homologous to a mouse isoleucine tRNA. The element forms a repeated family in the human genome that I name RTVL-I (retrovirus-like element-isoleucine). In the coding region of the Hpr gene, there are no frameshift or nonsense mutations and its exon-intron splicing sites, 5' flanking and 3' flanking sequences do not show any obvious defects. There are 28 amino acid differences between the decoded amino acid sequences of the Hpr and Hp genes. Sixteen of these differences occur in the hpr beta chain, and all appear to be located on the surface of the molecule in places not thought to be involved in the hemoglobin binding function of haptoglobin. The structure of the Hpr gene suggests that the gene may be expressed and give rise to a functional product.     

The transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein

The synthesis of a variety of proteins, including the well characterized degradative enzymes, which occurs during the transition state between vegetative growth and the onset of sporulation in Bacillus subtilis is controlled by a class of molecules known as transition state regulators. One of these regulators is the product of the hpr gene, first identified by mutations affecting the synthesis of extracellular proteases. We have purified the Hpr protein and found that it binds specifically to DNA fragments carrying the promoters and the upstream regions of the alkaline (aprE) and neutral (nprE) protease genes of B. subtilis. DNase I protection experiments revealed that the Hpr protein is able to bind at four and two regions of the aprE and nprE promoters, respectively. We have also located two Hpr binding sites in the promoter region of a gene of unknown function which is nevertheless known to be developmentally regulated during the transition state and which occurs in the same operon as the gene encoding another transition state regulator, Sin. The location of one of the Hpr binding sites on the aprE gene occurs adjacent to a region to which the Sin protein binds. However, in mixing competition experiments we have shown that Hpr and Sin binding occurred independently, and no visible alterations of protected regions were detected.     

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene

The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined. The ptsH promoter was localized using the S1 mapping technique. A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter. The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIGlc of the PTS). Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.     

Structure and expression of the human haptoglobin locus.

Human genomic clones of the haptoglobin Hp1F and the "haptoglobin related' gene (Hpr) have been isolated. The two genes are adjacent, spanning a region of approximately 21 kb. A comparison of their coding sequences shows that Hpr differs from Hp1F at 28 codons. Northern blot and primer elongation analyses with human liver RNA show that the haptoglobin gene Hp1F appears to be transcribed some 1000-fold less in fetal than in adult liver. In adult liver the amount of Hpr mRNA is at the lower limit of detection, therefore the extent of its expression is at most less than 1000-fold that of the Hp1F gene. No Hpr mRNA can be detected in fetal liver.     

Biochemical properties and phylogeny of hydroxypyruvate reductases from methanotrophic bacteria with different c<Subscript>1</Subscript>-assimilation pathways

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C₁-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C₁-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.     

The molecular basis of sulfonylurea herbicide resistance in tobacco

The enzyme acetolactate synthase (ALS) is the target enzyme for the sulfonylurea and imidazolinone herbicides. We describe the isolation and characterization of the ALS genes from two herbicide-resistant mutants, C3 and S4-Hra, of Nicotiana tabacum. There are two distinct ALS genes in tobacco which are 0.7% divergent at the amino acid sequence level. The C3 mutant has a single Pro-Gln replacement at amino acid 196 in one ALS gene. This gene is termed the class I gene and is equivalent to the SuRA locus. The S4-Hra mutant has two amino acid changes in the other ALS gene. This gene is termed the class II gene or the SuRB locus. The S4-Hra mutant includes a Pro-Ala substitution at amino acid 196 and a Trp-Leu substitution at amino acid 573. Gene reintroduction experiments have confirmed that these amino acid substitutions are responsible for the herbicide resistance phenotypes. Transgenic plants carrying these genes are highly resistant to sulfonylurea herbicide applications.     

Expression of the Cucumber Hydroxypyruvate Reductase Gene Is Down-Regulated by Elevated CO2

We examined the effects of CO2 concentration on the white-light-stimulated expression of the cucumber (Cucumis sativus L.) Hpr gene. Hpr encodes hydroxypyruvate reductase, an enzyme important in the photorespiratory glycolate pathway, which plays an integral role in carbon allocation in C3 plants. Because CO2 is an end product of this pathway and because increased CO2 concentrations lessen the need for photorespiration, we tested whether exposure of plants to elevated CO2 would affect white-light-stimulated Hpr gene expression. Exposure of dark-adapted cucumber seedlings to elevated CO2 (2 to 3 times ambient) during a 4-h white-light irradiation significantly inhibited the accumulation of Hpr mRNA. Increasing the CO2 concentration during irradiation to 6 or 9 times ambient did not further inhibit Hpr mRNA accumulation. The depressing effect of high CO2 on Hpr mRNA accumulation was seen in both high and low light, but was more pronounced in higher light. These results suggest that maximum sensitivity to CO2 occurs in conditions near those normally encountered by the plant (high light, CO2 concentration near ambient) and support a model in which white-light-regulated Hpr expression is modulated in part by environmental CO2 concentration.     

DNase I hypersensitivity in the gamma globin gene locus of K562 cells.

We have probed the chromatin conformation of the G gamma-A gamma-delta-beta globin gene locus of K562 cells, a human hematopoietic cell line, with the enzyme pancreatic DNAse I. This enzyme preferentially digests genes in an active configuration. We have found that in K562 cells, which produce embryonic and fetal but not adult hemoglobins, both the active gamma and inactive beta genes are DNAse I sensitive. However, only the active gamma genes have DNAse I hypersensitive regions. The hypersensitive regions have been mapped to an area approximately 100 base pairs 5' to the G gamma and A gamma genes.     

The trypanolytic factor of human serum: many ways to enter the parasite,a single way to kill

Humans have developed a particular innate immunity system against African trypanosomes, and only two Trypanosoma brucei clones (T. b. gambiense, T. b. rhodesiense) can resist this defence and cause sleeping sickness. The main players of this immunity are the primate‐specific apolipoprotein L‐I (apoL1) and haptoglobin‐related protein (Hpr). These proteins are both associated with two serum complexes, a minor subfraction of HDLs and an IgM/apolipoprotein A‐I (apoA1) complex, respectively, termed trypanosome lytic factor (TLF) 1 and TLF2. Although the two complexes appear to lyse trypanosomes by the same mechanism, they enter the parasite through various modes of uptake. In case of TLF1 one uptake process was characterized. When released in the circulation, haemoglobin (Hb) binds to Hpr, hence to TLF1. In turn the TLF1–Hpr–Hb complex binds to the trypanosome haptoglobin (Hp)–Hb receptor, whose original function is to ensure haem uptake for optimal growth of the parasite. This binding triggers efficient uptake of TLF1 and subsequent trypanosome lysis. While Hpr is involved as TLF ligand, the lytic activity is due to apoL1, a Bcl‐2‐like pore‐forming protein. We discuss the in vivo relevance of this uptake pathway in the context of other potentially redundant delivery routes.     

Non-random loss of uninterrupted ribosomal DNA repeating units upon induction of a bobbed mutation

To investigate the physical organization of ribosomal RNA genes of two bobbed (bb) loci carried by the Dp(1;f)122 free duplication, a wild type and a deleted one derived from it, genomic DNAs from XXNO-/Dp122bb+ and XXNO-/Dp122bb adult females were analyzed by restriction enzyme digestions. We found that in the bb mutant there was a loss of uninterrupted genes, while genes interrupted by type I and type II insertions remained apparently unchanged. This is an indication that at least in this wild type bb+ locus, carried by the 122 free duplication, the different repeating units are not distributed randomly. In fact, after digestion of the rDNA carried by the bb+ duplication with the enzyme BamHI that cuts only in type I insertions, we have obtained long uncleaved fragments of DNA containing uninterrupted genes.     

The role of enzyme I in the unmasking of an essential thiol of the membrane-bound enzyme II of the phosphoenolpyruvate-glucose phosphotransferase system of Escherichia coli.

The membrane-bound component of the phosphotransferase system of Escherichia coli, responsible for the phosphorylative uptake of methyl-alpha-D-glucoside has an essential thiol group which becomes available to inactivation by thiol reagents in the presence of the phosphate-accepting sugar or when phosphoenolpyruvate synthesis is inhibited. The form resistant to the thiol reagent requires not only the absence of sugar and an intact phosphoenolpyruvate generating system, but also an intact system generating phosphorylated Hpr which is impaired by heating of a thermosensitive enzyme I mutant.     

The role of enzyme I in the unmasking of an essential thiol of the membrane-bound enzyme II of the phosphoenolpyruvate-glucose phosphotransferase system of Escherichia coli

The membrane-bound component of the phosphotransferase system of Escherichia coli, responsible for the phosphorylative uptake of methyl-α-d-glucoside has an essential thiol group which becomes available to inactivation by thiol reagents in the presents of the phosphate-accepting sugar or when phosphoenolpyruvate synthesis is inhibited. The form resistant to the thiol reagent requires not only the absence of sugar and an intact phosphoenol-pyruvate generating system, but also an intact system generating phosphorylated Hpr which is impaired by heating of a thermosensitive enzyme I mutant.     

Mitochondrial malate dehydrogenase and malic enzyme: Mendelian inherited electrophoretic variants in the mouse

Malate dehydrogenase and malic enzyme each possess supernatant and mitochondrial molecular forms which are structurally and genetically independent. We describe electrophoretic variants of the mitochondrial enzymes of malate dehydrogenase and malic enzyme in mice. Progeny testing from genetic crosses indicated that the genes which code for mitochondrial malate dehydrogenase and malic enzyme were not inherited maternally but as independent unlinked nuclear autosomal genes. The locus for mitochondrial malic enzyme was located on linkage group I. Linkage analysis with a third mitochondrial enzyme marker, glutamic oxaloacetic transaminase, showed that the nuclear genes which code for the three mitochondrial enzymes were not closely linked to each other. This evidence suggests that clusters of nuclear genes coding for mitochondrial function are unlikely in mice.Supported by U.S. Public Health Service grants 5F2 HD-35,531 and GM-09966.     

Isolation and analysis of the genes for cytochrome c oxidase in Paracoccus denitrificans

Synthetic oligonucleotide probes were used to clone two loci from the chromosomal DNA of Paracoccus denitrificans that contain the genes for cytochrome c oxidase (cytochrome aa₃). One locus seems to contain four or five genes probably forming an operon. Two of these code for the oxidase subunits II and III. Three open reading frames are found between the COII and COIII genes. The other locus codes for the subunit I. A short open reading frame is found upstream of this gene. All three subunits of the Paracoccus enzyme show remarkable homology to the corresponding subunits of the mitochondrial cytochrome oxidase. Possible protein products of the open reading frames have not yet been identified.     

Chromosomal assignment of the alcohol dehydrogenase cluster locus to human chromosome 4q21-23 by in situ hybridization

Human class I alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1; ADH) is the major enzyme involved in ethanol oxidation. Three highly homologous genes govern the synthesis of three types of subunits which form several ADH isozymes. The locus for class I ADH loci was previously assigned to q21-25 of chromosome 4 by somatic cell hybridization techniques. Analysis of grain positions by in situ hybridization of chromosomes indicated that the ADH cluster locus is on 4q21-23, probably 4q22.