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PubMed1993

cDNA cloning of a germ cell specific lamin B3 from mouse spermatocytes and analysis of its function by ectopic expression in somatic cells.

The nuclear lamina is a fundamental component involved in the assembly of the nuclear envelope and higher order chromosomal structures in eukaryotes. In mammals, it is composed of four major lamin proteins, termed lamins A, B1, B2 and C. Here we first report cDNA cloning of a new 53 kDa lamin protein from mouse spermatocytes, termed lamin B3, the expression of which appears restricted to spermatogenic cells. Its gene structure indicates that lamin B3 is generated by differential splicing and alternative polyadenylation from lamin B2. When lamin B3 is introduced into somatic cells in culture, their nuclear morphology is transformed from spherical to hook-shaped. On the basis of the results obtained, we suggest that the germ cell specific lamin B3 is involved in the reorganization of nuclear and chromosomal structures during meiotic division.

Aging↗

PubMedDate not supplied

[Structure of chromosomal deoxyribonucleoproteins. X. Investigation of protein organization in chromosomal subunits with the aid of bifunctional agents].

Chromosomal proteins were treated with imido esters or with formaldehyde. Histones and their oligomers were then obtained by acid extraction and analyzed by two-dimensional gel electrophoresis. We discovered a nonameric histone oligomer in which histone H1 complexes with the octamer of small histones. In this complex, H1 interacts preferentially with H2a and H3. One can suppose from these experiments that histone H1 has close contacts with core histones. Another conclusion from the results obtained is that the structure of the nucleosome core is different in H1-containing mononucleosomes and in core particles. Thus, histone H1 is an important component of the nucleosomal protein complex and its presence is necessary for supporting the native compact state of the nucleosomal core.

Animals↗

PubMed1993

Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

Alcohol Dehydrogenase↗

PubMed1999

Genomic structure and chromosomal localization of the mouse Hsf2 gene and promoter sequences.

The mouse heat shock factor 2 (HSF2) cDNA was previously cloned by homology to HSF1, the heat shock factor involved in the cellular response to stress [Sarge, K.D., Zimarino, V., Holm, K., Wu, C., Morimoto, R.I., Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. Genes Dev. 5 (1991) 1902-1911]. HSF2 is active in restricted cell types during pre- and post-implantation stages of development, and only in male germ cells of adult mice. However, the function of this factor remains elusive. We report here the cloning of the mouse Hsf2 gene and its genomic structure. We show that the gene is composed of 13 exons of variable sizes spanning at least 43kb in the genome. The transcription start site has been determined, and upstream sequences with promoter activity have been identified by their ability to direct the expression of a luciferase reporter gene in transfected cells. A preliminary analysis of the proximal promoter sequence determined that the TATA box is absent, but that a GC-rich region with several potential binding sites for transcription factors is present. The gene has been mapped to mouse chromosome 10 by in-situ hybridization on metaphase chromosomes.

Animals↗

PubMed1994

Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene.

Nitric oxide, a multifunctional effector molecule synthesized by nitric oxide synthase (NOS) from L-arginine, conveys signals for vasorelaxation, neurotransmission, and cytotoxicity. Three different NOS isoforms have been identified which fall into two distinct types, constitutive and inducible. The inducible NOS (iNOS) isoform is expressed in a variety of cell types and tissues in response to inflammatory agents and cytokines. The human iNOS (NOS2) gene was isolated on overlapping cosmid clones from a human genomic library using both the murine macrophage and the human hepatocyte iNOS cDNAs as probes. All isolated cosmids were part of a single genomic locus and no other genomic loci were identified or isolated. Analysis of this locus indicated that the human iNOS gene is approximately 37 kilobases in length and consists of 26 exons and 25 introns. Primer extension analysis of lipopolysaccharide and cytokine-stimulated human hepatocyte RNA mapped the transcriptional initiation site 30 base pairs downstream of a TATA sequence, and a 400-base pair 5'-flanking region was found to be structurally similar to the recently described murine iNOS promoter. Polymerase chain reaction analysis of a human/rodent genomic DNA somatic cell hybrid panel and fluorescent in situ hybridization indicated that the human iNOS gene is located on chromosome 17 at position 17cen-q11.2.

Amino Acid Oxidoreductases↗

PubMed2003

[Detection of 29 types of fusion gene in leukemia by multiplex RT-PCR].

OBJECTIVE: To analyze the fusion genes derived from 29 types of chromosome structural aberrations in children with leukemia. METHODS: Bone marrow samples from 191 children with leukemia were analyzed with a novel multiplex nested RT-PCR. RESULTS: Of the 191 leukemic samples, 86 (45.0%) carried 14 types of fusion genes including SIL/TAL1, MLL/AF1q, E2A/PBX1, MLL/AF6, AML1/ETO, MLL/AF9, TEL/ABL, BCR/ABL, MLL/AF10, dupMLL, MLL/ENL, TEL/AML1, PML/RARalpha and CBFbeta/MYH11. The activation of oncogene HOX11 was detected in 31 cases, with or without other chromosome aberrations in 15 (7.8%) and 16 cases (8.4%), respectively. CONCLUSION: This multiplex nested RT-PCR reaction could screen 29 types of chromosome structural aberrations at the same time. It may be helpful for the detection of minimal residual diseases after chemotherapy and bone marrow transplantation.

Adolescent↗

PubMedDate not supplied

[Structure of chromosomal deoxyribonucleoproteins. VII. Free dna in preparation of fragmented chromatin].

Chromatin which was hydrodynamically sheared in a low ionic strength buffer lacking divalent cations (mu = 0.005) contains a heterogeneous set of DNP particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1-2 mM MgCl2 or to 0.1-0.2M NaCl results in the appearance of completely free DNA molecules. A salt-induced rearrangementof DNA-bound histones, but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining DNP particles. Percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of approx. 400 base pairs is increased from zero in the initial DNP sample to 8-9% in 1 mM MgCl2 and further to 30-31% of the total DNA in 0.30 M NaCl, 2 mM MgCl2. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde-fixed DNP in CsCl gradients but also in non-ionic Metrizamide gradients with either fixed or unfixed DNP samples. Process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium. Percentage of free DNA molecules in the sheared chromatin at a given ionic strength of solution is strongly decreased upon an increase of the average length of DNA in the DNP particles. Several lines of evidence suggest that free DNA molecules are formed in the sheared chromatin as a result of cooperative rearrangements of histones in salt-induced DNP aggregates. A dynamical model of chromosomal fiber is proposed on the basis of the present and earlier experimental data [1]. According to the model histones are arranged on DNA in clusters separated by stretches of free DNA. A salt-induced migration of histones along or between DNP fibers can result in unification of different clusters, thereby generating longer stretches of free DNA, the total amount of free DNA being approximately constant. Possible in vivo significance of such a dynamical structure is discussed.

Animals↗

PubMed2001

Chromosomal localization, structure, single-nucleotide polymorphisms, and expression of the human H-protein gene of the glycine cleavage system (GCSH), a candidate gene for nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is an inborn error of metabolism caused by deficiency in the glycine cleavage system (GCS); this system consists of four individual constituents, P-, T-, H-, and L-proteins. Several mutations have been identified in P- and T-protein genes, but not in the H-protein gene (GCSH), despite the presence of case reports of H-protein deficiency. To facilitate the mutational and functional analyses of GCSH, we isolated and characterized a human p1-derived artificial chromosome (PAC) clone encoding GCSH. GCSH spanned 13.5kb and consisted of five exons. Using the PAC clone as a probe, we mapped GCSH to chromosome 16q24 by fluorescence in situ hybridization. The transcription initiation site was determined by the oligonucleotide-cap method, and potential binding sites for several transcriptional factors were found in the 5' upstream region. Direct sequencing analysis revealed five single-nucleotide polymorphisms. The expression profiles of P-, T-, and H-protein mRNAs were studied by dot-blot analysis, using total RNA from various human tissues. GCSH was expressed in all 29 tissues examined, while T-protein mRNA was detected in 27 of the 29 tissues. In contrast, the P-protein gene was expressed in a limited number of tissues, such as liver, kidney, brain, pituitary gland, and thyroid gland, suggesting distinct transcriptional regulation of each GCS constituent.

Alleles↗

PubMed1988

cDNA, deduced polypeptide structure and chromosomal assignment of human pulmonary surfactant proteolipid, SPL(pVal).

In hyaline membrane disease of premature infants, lack of surfactant leads to pulmonary atelectasis and respiratory distress. Hydrophobic surfactant proteins of Mr = 5,000-14,000 have been isolated from mammalian surfactants which enhance the rate of spreading and the surface tension lowering properties of phospholipids during dynamic compression. We have characterized the amino-terminal amino acid sequence of pulmonary proteolipids from ether/ethanol extracts of bovine, canine, and human surfactant. Two distinct peptides were identified and termed SPL(pVal) and SPL(Phe). An oligonucleotide probe based on the valine-rich amino-terminal amino acid sequence of SPL(pVal) was utilized to isolate cDNA and genomic DNA encoding the human protein, termed surfactant proteolipid SPL(pVal) on the basis of its unique polyvaline domain. The primary structure of a precursor protein of 20,870 daltons, containing the SPL(pVal) peptide, was deduced from the nucleotide sequence of the cDNAs. Hybrid-arrested translation and immunoprecipitation of labeled translation products of human mRNA demonstrated an Mr = 22,000 precursor protein, the active hydrophobic peptide being produced by proteolytic processing to Mr = 5,000-6,000. Two classes of cDNAs encoding SPL(pVal) were identified. mRNA of approximately 900 bases was identified on Northern analysis of fetal and adult RNA. Human SPL(pVal) mRNA was more abundant in the adult than in fetal lung. The SPL(pVal) gene locus was assigned to chromosome 8.

Amino Acid Sequence↗

PubMed1997

Chromosomal localization, structure, and regulation of the UGT2B17 gene, encoding a C19 steroid metabolizing enzyme.

UGT2B17 is a UDP-glucuronosyltransferase enzyme expressed in several extrahepatic steroid target tissues, including the human prostate, where it glucuronidates C19 steroids such as dihydrotestosterone (DHT), androsterone (ADT), and androstane-3alpha, 17beta-diol (3alpha-diol). To determine if UGT2B17 is regulated by physiological effectors of the human prostate, DHT and epidermal growth factor (EGF) were demonstrated to specifically down-regulate the steady-state levels of UGT2B17 transcript and protein in LNCaP cells (Guillemette et al., 1997). These results implicate regulation of UGT2B17 at the level of gene transcription, therefore, a P-1-derived artificial chromosome (PAC) clone of 120 kb containing the entire UGT2B17 gene was isolated. The gene is comprised of six exons spanning approximately 30 kb, and fluorescence in situ hybridization of the UGT2B17 PAC clone to normal human lymphocyte chromosomes, mapped the gene to chromosome 4q13. To determine if the 5'-flanking DNA of the UGT2B17 gene is sufficient to confer gene expression, a 2,942-bp fragment was subcloned into a luciferase reporter plasmid and yielded an activity of 25-fold over background when transfected in LNCaP cells. However, transfection of the construct into HK-293, MCF-7, JEG-3, and HepG2 cells yielded only a moderate activity of two- to five-fold over background. Treatment of transfected LNCaP cells with 10 nM R1881, a nonmetabolizable analog of DHT, and 10 ng/ml EGF decreased the luciferase activity by 60%. This suggests that at least part, if not all, of the inhibitory effect of EGF and DHT on UGT2B17 is at the level of transcription. Progressive 5' deletions of the UGT2B17 5'-flanking region in the luciferase constructs alleviated the inhibition by R1881 and EGF, and revealed several potential responsive elements that may confer the observed regulation of the UGT2B17 gene. This study demonstrates regulation of the UGT2B17 gene by physiological effectors of the human prostate and supports the hypothesis that UGT enzymes are involved in steroid metabolism in extrahepatic tissues.

Amino Acid Sequence↗

PubMed1994

Clastogenicity of triazinoindole derivatives in relation with the physicochemical properties of the substituents.

The clastogenicity of four compounds of the series 3-(4'-substituted-benzylidenamino)5H-1,2,3-triazin [5,4-b]indol-4-one, which showed an important activity as inhibitors of platelet aggregation, has been evaluated. The compounds studied differed in the physicochemical properties of the substituent occupying the 4' position of the benzilidenamino group. The 4' substituents were: -H, -NO2, -OCH3 and -C6H5. They were tested on V-79 cells, both with and without metabolic activation, and structural chromosome aberrations were scored. Compounds with -H, -NO2 and -OCH3 radicals were active both with and without metabolic activation. Compound with -C6H5 radical was clastogenic only at the highest dose tested and with metabolic activation. It appears that the compound with the biggest and most hydrophobic substituent is the least clastogenic of the series. These results are in agreement with some previous ones obtained in bacteria and show a good correlation between the results of the Ames test and the structural chromosome aberrations test.

Bacteria↗

PubMed1992

Differentiated recurrence risk estimations in the Prader-Willi syndrome.

The Prader-Willi syndrome usually occurs sporadically. About 60% of the patients show a chromosomal deletion of proximal 15q. There are only a few reports on familial recurrence. Two previous estimates give either a low but recognizable recurrence risk of 1.6% or a risk of less than 0.1%. The inconsistency is obviously due to differences in the stringency of the diagnostic criteria used and to different modes of ascertainment of cases. This gave rise to a review of the literature, leading to the following suggestions: (1) A deletion at 15q has not been found in a familial case of PWS, except in those cases where del(15q) is due to familial structural chromosome rearrangements. Hence, with de novo deletion, the recurrence risk should be nearly zero. (2) In cases with familial translocation, risk estimates depend on the variable nature of the familial chromosome mutation. (3) If only one child is affected and has no deletion at 15q, this may be a sporadic or an isolated familial case. For this situation I estimated an overall recurrence risk of 0.4%. (4) If two or more sibs are affected, I consider a risk of 50% that the next sib may also be affected. This estimate reflects the observed segregation ratio and is in accordance with the proposed genomic imprinting model in the etiology of PWS. Prenatal cytogenetic diagnosis in a pregnancy at risk for PWS is hampered since del(15q) is almost exclusively found in sporadic cases. Hence, this method is limited to those very rare cases where del(15q) is due to familial structural chromosome rearrangements.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Aberrations↗

PubMed2001

Genotoxic effects of radiotherapy and chemotherapy on circulating lymphocytes in patients with Hodgkin's disease.

PURPOSE: The aim of this study was to find out the structural chromosomal changes in somatic cells after chemotherapy (CT) with or without radiotherapy (RT). METHODS AND MATERIALS: This prospective study included 30 Hodgkin's disease (HD) patients. The patients of Group I(1) had only MOPP/ABV CT. The patients of Group II(2) also had irradiation. Group III(3) (control group) consisted of healthy subjects without any reported malignant disease. Mutagenetic testing was performed at the time of diagnosis and was repeated immediately after treatment and again 6 months later. The following tests were applied: structural chromosomal aberrations (CA), sister chromatid exchange (SCE) and micronucleus (MN) tests. RESULTS: Prior to treatment, the chromosome damage in our patients was not higher than that in the control group. Immediately after the complete treatment, we observed a strong inhibition of the mitotic activity of lymphocytes as well as a significant increase in the frequency of CA, MN and SCE in the Groups I and II. In patients treated by RT, we found statistically significant differences between the Groups I and II in MN (P<0.005) and CA frequencies (P<0.005), and an increased number of dicentrics (P=0.021). Six months after the complete treatment, the mitotic activity was found to be nearly normal, but chromosome damage occurred. CA and SCE values did not differ much from the values measured immediately after treatment, whereas MN values decreased without returning to the baseline levels. The chromosome damage persisted even 6 months after combined RT and CT. The damage in the genome of individual cells was in some cases even greater than immediately after treatment. The possible risk of neoplastic transformation posed by these heavily damaged cells, if viable, due to the changes in the expression of oncogenes or tumour suppresser genes, is discussed.

Adolescent↗

PubMed1999

Phosphorylated proteins are involved in sister-chromatid arm cohesion during meiosis I.

Sister-chromatid arm cohesion is lost during the metaphase I/anaphase I transition to allow homologue separation. To obtain needed information on this process we have analysed in grasshopper bivalents the sequential release of arm cohesion in relation to the behaviour of chromatid axes. Results show that sister axes are associated during early metaphase I but separate during late metaphase I leading to a concomitant change of chromosome structure that implies the loss of sister-kinetochore cohesion. Afterwards, homologues initiate their separation asynchronously depending on their size, and number and position of chiasmata. In all bivalents thin chromatin strands at the telomeres appeared as the last point of contact between sister chromatids. Additionally, we have analysed the participation of phosphoproteins recognised by the MPM-2 monoclonal antibody against mitotic phosphoproteins in arm cohesion in bivalents and two different kinds of univalents. Results show the absence of MPM-2 phosphoproteins at the interchromatid domain in mitotic chromosomes and meiotic univalents, but their presence in metaphase I bivalents. These phosphoproteins are lost at the onset of anaphase I. Taken together, these data have prompted us to propose a 'working' model for the release of arm cohesion during meiosis I. The model suggests that MPM-2 phosphoproteins may act as cohesive proteins associating sister axes. Their modification, once all bivalents are correctly aligned at the metaphase plate, would trigger a change of chromosome structure and the sequential release of sister-kinetochore, arm, and telomere cohesions.

Anaphase↗

PubMed2005

[Attitude of pregnant women towards prenatal screening for chromosomal and structural fetal anomalies between 11-14 weeks of gestation].

AIM: The aim of the present survey is to analyze the attitude of pregnant women towards the possibilities of prenatal screening and diagnosis between 11-14 weeks of gestation (w.g.). MATERIAL AND METHODS: Overall 109 pregnant women, hospitalized in SBALAG "Maichin dom", received written information about the possibilities for prenatal screening and diagnosis between 11-14 w.g. and 16-20 w.g., as well as a comprehensive description of the potential advantages and disadvantages of the different approaches/methods of screening/diagnosis. Consequently, the patients were asked to answer 13 closed and semi-closed questions in an anonymous enquiry. RESULTS: Overall 97,2% (106/109) of the women agreed to perform an ultrasound examination for fetal nuchal translucency measurement as a screening tool for chromosomal anomalies between 11-14 w.g. Furthermore, 82,6% (90/109) preferred the option of first trimester biochemical screening, rather than screening in the second trimester. On the other hand only 62,4% (68/109) accepted chorionic villous sampling between 11-14 w.g. in comparison to 83,5% (91/109) who agreed to perform an amniocentesis between 16-20 w.g. CONCLUSION: The vast majority of women prefer prenatal screening programs to be scheduled in the first, rather than in the second trimester. However, most women would still choose for a relatively safer second trimester invasive procedure instead of the option of earlier prenatal diagnosis.

Attitude to Health↗

PubMed1992

Abnormal regional hypermethylation in cancer cells.

Let me summarize by reviewing a model which is meant to raise as many questions as it answers (Fig. 2). What I have discussed today are data suggesting that during progression of solid tumors, like colon cancer, an increased cellular DNA methylating capacity characterizes the initial stages of multi-clonal hyperplasia. Despite this increase, the altered pattern of DNA methylation which subsequently emerges is largely manifest by a widespread hypomethylation of DNA. However, on a more regional basis, areas of hypermethylation appear which can affect strategic areas such as normally unmethylated CpG islands. These shifted DNA methylation patterns have the capacity to both follow, or cause, chromatin changes that can both directly silence genes critical for normal cell maturation--and/or participate in the structural chromosome changes which constitute genetic instability during tumor progression (Fig. 2). I suggest that one must view these changes as an interchangeable cycle of events during tumor progression. The chromatin changes and abnormal methylation patterns can drive one another with increasingly deleterious effects as the malignant phenotype emerges (reviewed in Baylin, 1991). What are the molecular events that would initiate the above dynamics? A working construct model is shown in Fig. 3. As discussed for the normal adult cell, there is a delicate balance between the strategic location of DNA MTase, regulation of this enzyme, and rate of DNA synthesis at replication forks (top panel, Fig. 3). In pre-neoplastic and cancer cells, perhaps failure of cells to exit the cell cycle and halt DNA replication, facilitates some sort of pressure to increase cellular DNA methyltransferase activity (bottom panel, Fig. 3). This increase may involve loss of feedback inhibition of the enzyme during the post DNA replication phase. There are also probable structural alterations in the nucleus which may alter the geographic relationship between the DNA replication fork and DNA MTase. In consequence, many DNA areas that should be getting methylated do not, and novel areas of methylation also arise. This cycle of events leads to the imbalance of DNA methylation that I have talked about. Future investigations of these possibilities, and of their specific consequences for alterations of gene expression and chromosome structure, may reveal a key molecular step underlying virtually all stages of tumor progression.

Adult↗

PubMed1988

The effects of in vivo administration of teratogenic doses of vitamin A during the preimplantation period in the mouse.

To examine the effects of vitamin A administered during the preimplantation period, pregnant C3H mice were exposed to teratogenic doses of the vitamin 60 h after copulation. Fetuses were examined for gross abnormalities on the 18th day of gestation and viability, cell number, mitotic index, and chromosome structure were assessed in 81-h blastocysts to determine whether embryotoxic effects were apparent in the preimplantation embryo. There was a reduction in the fetal weight of 18-day fetuses treated in this manner with 15,000 and 30,000 IU vitamin A (p less than 0.0003 in each case), and doses of 10,000 IU and greater were associated with a significantly higher incidence of gross abnormalities. Malformations included exophthalmos, anophthalmia, microphthalmia, exencephaly, exomphalos, and limb defects. Administration of 30,000 IU vitamin A resulted in resorption and intrauterine death in 70% of cases. There was no indication that vitamin A adversely affected 81-h blastocyst viability, cell number, mitotic index, and chromosome structure. The findings suggest that the teratogenic effects that were noted later in fetal life were the result of an action on the developing fetus of the vitamin at a stage later than 81-h and are consistent with the relative resistance of the preimplantation embryo to toxic injury. Persistence of vitamin A, either in the mother or the embryo, is the most likely explanation for the later expression of toxic injury, which is characteristic of the effects that are noted as a result of exposure to the teratogen during the period of organogenesis.

Abnormalities, Drug-Induced↗

PubMed1994

cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases.

Two types of 12-lipoxygenase that catalyze the transformation of arachidonic acid to 12(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) have been previously classified into platelet-type and leukocyte-type categories. Here, we document, for the first time, a molecular characterization of both forms within the same species. The amino acid sequence of the murine platelet 12-lipoxygenase deduced from its cDNA is 58% identical to the murine spleen/leukocyte 12-lipoxygenase. Expression constructs carrying the cDNAs for the two 12-lipoxygenase forms were introduced into human embryonic kidney 293 cells. The platelet-type enzyme metabolized arachidonic acid exclusively to 12-HPETE, whereas the leukocyte-type enzyme formed both 12-HPETE and 15-hydro(pero)xyeicosatetraenoic acid in a ratio of approximately 3:1. Linoleic acid was metabolized to a similar extent by the latter enzyme to 13-hydro(pero)xyoctadecadienoic acid but not by the platelet enzyme. Mutagenesis and deletion of the highly conserved lipoxygenase C-terminal isoleucine (Ile663), a residue believed to be involved in the non-heme iron atom coordination of all lipoxygenases, was performed. Deletion of Ile663 and substitution with most amino acids abolished enzyme activity. Only a valine substitution retained significant activity. These findings would tend to indicate a stringent requirement for the proper spatial alignment and folding of the C-terminal chain back into the core of the enzyme to interact with the iron atom by analogy with the recently determined crystal structure of a soybean lipoxygenase (Boyington, J. C., Gaffney, B. J., and Amzel, L. M. (1993) Science 260, 1482-1486). The platelet-type and leukocyte-type 12-lipoxygenase genes were cloned from a murine 129 Sv genomic library. Both genes are divided into a similar 14-exon/13-intron format, with the platelet-type gene being approximately twice the size of the leukocyte-type gene (13 versus 7.5 kilobases). A segment of a third gene was also isolated and probably represents a pseudogene derivative of either of these 12-lipoxygenase genes. All three genes were mapped to the central region of mouse chromosome 11 in a region of homology with human chromosome 17. Antibodies prepared against the two forms of 12-lipoxygenase revealed the differential distribution of the two enzymes throughout the mouse.

Amino Acid Sequence↗