A tissue-specific decrease in the pre-mRNA level of L1-and Alu-containing alleles of human genes

LINE1 and Alu retroelements occupy approximately 17 and 13% of the human genome, respectively. They include the evolutionarily youngest element groups Ta-L1, AluYa5, and AluYb8, many inserts of which are polymorphic in the Homo sapiens population. Despite the data on the ability of L1 and Alu elements to cause various modifications of the genome, the effects of these retroelements on gene expression have yet not been studied. Using the RT PCR method, we analyzed the pre-mRNA (heterogeneous nuclear RNA) content of allele pairs of four genes in five human cell lines, heterozygous with respect to intronic inserts of L1 and Alu elements. We showed for the first time a tissue-specific decrease in the pre-mRNA content of the gene allele bearing L1 or Alu inserts relative to the other allele of the same gene lacking the retroelement.     

The human episome HALF1: Structure of its genomic counterpart

A human episomal sequence (HALF1) has been identified by its ability to restore expression of hepatic functions when used to transfect a rat dedifferentiated cell line. The genomic equivalent of this human episome (gHALF1) and its flanking sequences were analyzed. HALF1 itself does not present the characteristics of a transposable element but half of its sequence corresponds to retroposons, including Alu and L1 repeats and a processed pseudogene, known to transposevia RNA intermediates. The structural characteristics of these different kinds of retroposons and their origin and evolution were analyzed.     

Isolation and cloning by a polymerase chain reaction of a genomic DNA fragment of the human slow skeletal troponin (TNNT1) gene

The genomic 3′ structure of the gene coding for the human slow skeletal troponin T (TNNT1) gene, is reported. An intron of 912 nucleotides containing an Alu-element has been identified and characterized. The complexity of the sequenced region suggests an alternative exon use. The present results may be valuable for further studies on the gene structure of TNNT1 and the related troponin gene family.     

Cloning, characterization and genomic organization of LCC-1 (scya16), a novel human CC chemokine expressed in liver

By homology search of expressed sequence tags (EST) in GenBank a novel member of the CC chemokine family was identified. The full-length sequence of this liver-specific CC chemokine (LCC-1) predicted a mature protein of 97 amino acids with 31-48% identity to other CC chemokines. There was a characteristic amino acid C-term extension when aligned with other chemokines. Northern blot analysis from a panel of human tissues revealed that LCC-1 mRNA expression is restricted to adult and fetal liver. Different polyadenylation results in two mRNA species of 1.5 kb and 0.5 kb in size. LCC-1 is constitutively expressed in human HepG2 hepatoma cells and is induced by hypoxic exposure. The promoter region of the LCC-1 gene contains potential HIF-1 binding sites. The EST for LCC-1 has been previously mapped to the CC chemokine cluster on human chromosome 17q11.2. The organization of the LCC-1 gene (scya16) into three exons interrupted by two introns is identical to that found for other members of the CC chemokine family.     

Transfer RNA-like structure of the human Alu family: Implications of its generation mechanism and possible functions

Summary Structural resemblance of the human Alu family with a subset of vertebrate tRNAs was detected. Of four tRNAs, tRNA^Lys, tRNA^Ile, tRNA^Thr, and tRNA^Tyr, which comprise a structurally related family, tRNA^Lys is the most similar to the human Alu family. Of the 76 nucleotides in lysine tRNA (including the CCA tail), 47 are similar to the human Alu family (60% identity). The secondary structure of the human Alu family corresponding to the D-stem and anticodon stem regions of the tRNA appears to be very stable. The 7SL RNA, which is a progenitor of the human Alu family, is less similar to lysine tRNA (55% identity), and the secondary structure of the 7SL RNA folded like a tRNA is less stable than that of the human Alu family folded likewise. Insertion of the tetranucleotide GAGA, which is an important region of the second promoter for RNA polymerase III in the Alu sequence, occurred during the deletion and ligation process to generate the Alu sequence from the parental 7SL RNA. These results suggest that the human Alu family was generated from the 7SL RNA by deletion, insertion, and mutations, which thus modified the ancestral 7SL sequence so that it could form a structure more closely resembling lysine tRNA. The similarities of several short interspersed sequences to the lysine tRNA were also examined. TheGalago type 2 family, which was reported to be derived from a methionine initiator tRNA, was also found to be similar to the lysine tRNA. Thus lysine tRNA-like structures are widespread in genomes in the animal kingdom. The implications of these findings in relation to the mechanism of generation of the human Alu family and its possible functions are discussed.     

Variations in the organization of human genomic DNA segments containing H1 histone genes

We have isolated from a lambda Ch4A library four human genomic DNA segments containing H1 histone genes. Analysis of the representation and organization of histone coding sequences indicates that three of these cloned DNA segments contain both core and H1 histone genes. One of the cloned human H1 histone genes has no core histone genes in close proximity.     

A novel polymorphic Alu insertion embedded in a LINE 1 retrotransposon in the human X chromosome (DXS225): identification and worldwide population study

We describe a novel polymorphic Alu insertion (DXS225) on the human X chromosome (Xq21.3) embedded into an L1 retrotransposon. The DXS225 polymorphism was genotyped in 684 males from the CEPH Human Genome Diversity Panel. This insertion was found in all regions of the globe, suggesting that it took place before modern humans spread from Africa ca. 100,000 years ago. However, only one Amerindian population (Karitiana) showed this insertion allele, which may have been introduced by European admixture. Thus, it appears likely that the Alu insertion was absent from pre-Columbian America. Analysis of molecular variance worldwide demonstrated that 92.2% of the genetic variance was concentrated within populations. DXS225 is flanked by two microsatellites (DXS8114 and DXS1002), which are 86 kb apart and are in very strong linkage disequilibrium. The combination of a unique event polymorphism on the X chromosome in linkage disequilibrium with two rapidly evolving microsatellites should provide a useful tool for studies of human evolution.     

Poly(ADP-ribose)polymerase-1 is required for integration of the human immunodeficiency virus type 1 genome near centromeric alphoid DNA in human and murine cells

This study examined the efficiency of human immunodeficiency virus type 1 (HIV-1) integration in poly(ADP-ribose)polymerase-1 (PARP-1)-deficient murine cells and in human cell lines transfected with small interfering RNA against PARP-1 (PARP-1 siRNA). To semi-quantify the amount of integrated HIV-1 genome, real-time nested PCR was carried out using primers specific for Alu and alphoid DNA combined with primers for the HIV-1 genome. The results showed that the integration efficiency of the HIV-1 genome near Alu DNA, which is randomly distributed in the chromosome, is reduced in PARP-1-deficient murine cells, but not in PARP-1 siRNA-transfected human cells. By contrast, the integration efficiency of the HIV-1 genome near alphoid DNA, which is localized in the centromere region, is significantly reduced in PARP-1-deficient murine cells and in PARP-1 siRNA-transfected human cells. These results suggest that PARP-1 is required for HIV-1 integration near the centromere region both in human and murine cells.     

Subfamily structure and evolution of the human L1 family of repetive sequences

Summary Comparative analysis of the available 3′-portions of the human L1 (LINE-1) family of repeated sequences indicates that all the sequences can be classified in two major subfamilies. The division is based on patterns of diagnostic bases shared within L1 subfamilies of sequences but differing between them. The overall ratio of replacement to synonymous positions, occupied by the diagnostic bases in the large open reading frame of the L1 sequence, is 1.15. This indicates that both subfamilies were obtained from genes coding for functional proteins. The L1 subfamilies appear to be of different ages and may represent a “fossil record” of the same active gene at different times in the history of primates. The younger subfamily can be split further into at least two closely related branches of sequences. The above facts combined with the recent data for the Alu subfamily structure show that LINE and SINE families of interspersed repeats share discontinuous patterns in their evolution. These data are consistent with the model that both Alu and L1 families, as well as other pseudogene families, contain active genes producing discrete layers of pseudogenes throughout the history of primates. Models of evolutionary processes that could generate these discontinuities are discussed together with the possible biological role of Alu and L1 genes.     

Genomic organization and localization of the human CRMP-1 gene.

The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein considered to be involved in the collapsin-induced growth cone collapse during neural development. CRMP-1 belongs to the Unc-33 gene family. Here we report the genomic structure and the localization of the human CRMP-1 gene to chromosome 4p16.1. Sequence analysis revealed that the human CRMP-1 gene consists of 14 exons. We have also established sequencing assays for all its coding exons. This should permit the rapid screening for mutations to assess CRMP-1 role in genetic disorders mapped in the 4p16.1 region.     

Constitutive expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain

Cytochrome P-450 function as mono-oxygenases and metabolize xenobiotics. CYP1A1, a cytochrome P-450 enzyme, bioactivates polycyclic aromatic hydrocarbons to reactive metabolite(s) that bind to DNA and initiate carcinogenesis. Northern and immunoblot analyses revealed constitutive expression of Cyp1a1 and CYP1A1 in rat and human brain, respectively. CYP1A1 mRNA and protein were localized predominantly in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum and pyramidal neurons of CA1, CA2, and CA3 subfields of the hippocampus. RT-PCR analyses using RNA obtained from autopsy human brain samples demonstrated the presence of a splice variant having a deletion of 87 bp of exon 6. This splice variant was present in human brain, but not in the liver from the same individual, and was absent in rat brain and liver. Structural modeling indicated broadening of the substrate access channel in the brain variant. The study demonstrates the presence of a unique cytochrome P-450 enzyme in human brain that is generated by alternate splicing. The presence of distinct cytochrome P-450 enzymes in human brain that are different from well-characterized hepatic forms indicates that metabolism of xenobiotics including drugs could occur in brain by pathways different from those known to occur in liver.     

N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits

Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.     

The distributions of "new" and "old" Alu sequences in the human genome: the solution of a "mystery"

The distribution in the human genome of the largest family of mobile elements, the Alu sequences, has been investigated for the past 30 years, and the vast majority of Alu sequences were shown to have the highest density in GC-rich isochores. Ten years ago, it was discovered, however, that the small "youngest" (most recently transposed) Alu families had a strikingly different distribution compared with the "old" families. This raised the question as to how this change took place in evolution. We solved what was considered to be a "mystery" by 1) revisiting our previous results on the integration and stability of retroviral sequences, and 2) assessing the densities of acceptor sites TTTT/AA in isochore families. We could conclude 1) that the open state of chromatin structure plays a crucial role in allowing not only the initial integration of retroviral sequences but also that of the youngest Alu sequences, and 2) that the distribution of old Alus can be explained as due to Alu sequences being unstable in the GC-poor isochores but stable in the compositionally matching GC-rich isochores, again in line with what happens in the case of retroviral sequences.     

Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4

Summary. By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2. Received September 8, 2000 Accepted January 8, 2001