Identification of a wound-induced inhibitor of a nuclear factor that binds the carrot extensin gene

Following wounding of carrot (Daucus carota L.) roots, the activity of a nuclear factor (EGBF-1) that binds a 5′-region of the carrot extensin gene declines to undetectable levels within 48 h. Mixing of nuclear extracts from wounded roots with nuclear extracts from unwounded roots has demonstrated the existence of a wound-induced inhibitor of EGBF-1. Inhibition of EGBF-1 DNA-binding activity by nuclear extracts from wounded roots is shown to be specific for EGBF-1, and to be destroyed by heat treatment. In addition, inhibition is saturable and occurs rapidly. Active EGBF-1 can be reconstituted from its inhibited state by renaturation of proteins from mixed extracts following denaturation by boiling in sodium dodecyl sulfate and 2-mercaptoethanol, and electrophoretic separation, indicating that inhibition is dependent upon the reversible interaction of EGBF-1 with a titratable factor. However, EGBF-1 activity could not be detected in nuclear extracts from wounded roots following denaturation and electrophoretic separation. Inhibitory activity was not detectable in nuclear extracts from roots that had been trated with ethylene. The action of the inhibitor indicates one possible mechanism for the control of EGBF-1 activity in carrot roots following wounding.     

Genetic Peculiarity of the Yakut Population as Inferred from Autosomal Loci

The autosomal gene pool of Yakuts was analyzed with a panel of polymorphic Alu insertions. The observed allele frequencies were typical for other Asian ethnic groups. Genetic differentiation of three Yakut populations was relatively high, 2%. East Siberian ethnic groups were shown to have a common gene pool and to experience no intense gene flow from other populations. Development of the Yakut gene pool was assumed to involve no substantial genetic effect of neighboring populations. The results fit both autochthonous and southern origin hypotheses.     

Mono-allelic retrotransposon insertion addresses epigenetic transcriptional repression in human genome

Background

Retrotransposons have been extensively studied in plants and animals and have been shown to have an impact on human genome dynamics and evolution. Their ability to move within genomes gives retrotransposons to affect genome instability.

Methods

we examined the polymorphic inserted AluYa5, evolutionary young Alu, in the progesterone receptor gene to determine the effects of Alu insertion on molecular environment. We used mono-allelic inserted cell lines which carry both Alu-present and Alu-absent alleles. To determine the epigenetic change and gene expression, we performed restriction enzyme digestion, Pyrosequencing, and Chromatin Immunoprecipitation.

Results

We observed that the polymorphic insertion of evolutionally young Alu causes increasing levels of DNA methylation in the surrounding genomic area and generates inactive histone tail modifications. Consequently the Alu insertion deleteriously inactivates the neighboring gene expression.

Conclusion

The mono-allelic Alu insertion cell line clearly showed that polymorphic inserted repetitive elements cause the inactivation of neighboring gene expression, bringing aberrant epigenetic changes.     

Alu Repeats in the Human Genome

Highly repetitive DNA sequences account for more than 50% of the human genome. The L1 and Alu families harbor the most common mammalian long and short interspersed elements. An Alu element is a dimer of similar, but not identical, fragments of total size about 300 bp, and originates from the 7SL RNA gene. Each element contains a bipartite promoter for RNA polymerase III, a poly(A) tract located between the monomers, a 3"-terminal poly(A) tract, and numerous CpG islands, and is flanked by short direct repeats. Alu repeats constitute more than 10% of the human genome and are capable of retroposition. Possibly, these elements played an important part in genome evolution. Insertion of an Alu element into a functionally important genome region or other Alu-dependent alterations of gene functions cause various hereditary disorders and are probably associated with carcinogenesis. In total, 14 Alu families differing in diagnostic mutations are known. Some of these, which are present in the human genome, are polymorphic and relatively recently have been inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.     

Repetitive DNA sequences located in the central region of the human mdr1 (multidrug resistance) gene may account for a gene fusion event during its evolution

The mdr1 gene, first member of the human multidrug-resistance gene family, is a major gene involved in cellular resistance to several drugs used in anticancer chemotherapy. Its product, the drug-excreting P-glycoprotein, shows a bipartite structure formed by two similar adjacent halves. According to one hypothesis, the fusion of two related ancestral genes during evolution could have resulted in this structure. The DNA sequence analysis of the introns located in the region connecting the two halves of the human mdr1 gene revealed a highly conserved poly(CA) · poly (TG) sequence in intron 15 and repeated sequences of the Alu family in introns 14 and 17. These repeated sequences most likely represent molecular fossils of ancient DNA elements which were involved in such a recombination event. Correspondence to: M. Pauly     

人神经生长因子基因的克隆及序列分析

以人基因组DNA为模板,采用PCR获得人神经生长因子(hNGF)基因,载体pGEM-T中,DNA序列分析克隆的hNGF基因与文献报道的完全一致。     

人血小板因子4基因cDNA的克隆与序列测定

为研究人血小板因子4（hPF4)的生物学性能和用于肿瘤生物治疗的可能性,运用反转录聚合酶链反应（RT－PCR）从人工白血病细胞系HEL细胞总RNA中扩增出hPF4基因cDNA序列,将其克隆至pUC18载体中。序列分析证实克隆片段与献报道的hPF4基因cDNA序列完全一致,说明已成为克隆到hPF4基因cDNA。     

A possible hot spot for Ac insertion in the maize P gene.

Summary We have analyzed the footprints left by a single Ac transposable element during its intragenic transposition to different positions in the maize P gene. One site appears to have been visited twice by transposons, indicating that it may be an insertion hot spot. Implications of this finding for the origin of the P-vv allele are discussed. Analysis of transposon footprints may prove generally useful for establishing pedigree relationships among gene alleles.     

Expression, refolding, and characterization of a novel recombinant dual human stem cell factor

A novel recombinant dual human stem cell factor (rdhSCF) gene which consisted of a full-length hSCF(1-165aa) cDNA and a truncated hSCF (1-145aa) cDNA, linked by a peptide (GGGGSGGGGSGG) coding region, was constructed and cloned into Escherichia coli expression vector pET-22b. The rdhSCF was expressed at high level in E. coli BL21(DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in urea and refolded by ion-exchange chromatography. After renaturation, the purity of the yielded rdhSCF was up to 90%. Cell proliferation assay showed that the specific activity of the rdhSCF was 2.86x10(5) U/mg, about 1.66 times as high as that of monomer rhSCF expressed in E. coli.     

Local gene duplication with insertion of a complex microsatellite

A new gene,msta, was found in region 2E of theDrosophila melanogaster X-chromosome. The gene is expressed in the head of adults, consists of two exons, and codes for a protein containing the SET domain, as characteristic of several proteins modulating chromatin structure and gene activity. Its shortened copy,msts, was found in the vicinity of,msta. Since the divergence between their coding regions was lower than between the introns,msts was assumed to have functioned for some time after duplication. The genes proved to be separated by the 1.688-2E complex microsatellite.     

Investigation of the RH locus in gorillas and chimpanzees

The human Rh blood-group system is encoded by two homologous genes,RhD andRhCE. TheRH genes in gorillas and chimpanzees were investigated to delineate the phylogeny of the humanRH genes. Southern blot analysis with an exon 7-specific probe suggested that gorillas have more than twoRH genes, as has recently been reported for chimpanzees. Exon 7 was well conserved between humans, gorillas, and chimpanzees, although the exon 7 nucleotide sequences from gorillas were more similar to the humanD gene, whereas the nucleotide sequences of this exon in chimpanzees were more similar to the humanCE gene. The intron between exon 4 and exon 5 is polymorphic and can be used to distinguish the humanD gene from theCE gene. Nucleotide sequencing revealed that the basis for the intron polymorphism is anAlu element inCE which is not present in theD gene. Examination of gorilla and chimpanzee genomic DNA for this intron polymorphism demonstrated that theD intron was present in all the chimpanzees and in all but one gorilla. TheCE intron was found in three of six gorillas, but in none of the seven chimpanzees. Sequence data suggested that theAlu element might have previously been present in the chimpanzeeRH genes but was eliminated by excision or recombination. Conservation of theRhD gene was also apparent from the complete identity between the 3′-noncoding region of the human D cDNA and a gorilla genomic clone, including anAlu element which is present in both species. The data suggest that at least twoRH genes were present in a common ancestor of humans, chimpanzees, and gorillas, and that additionalRH gene duplication has taken place in gorillas and chimpanzees. TheRhCE gene appears to have diverged more thanRhD among primates. In addition, theRhD gene deletion associated with the Rh-negative phenotype in humans seems to have occurred after speciation. Correspondence to: C.M. Westhoff     

The use ofin situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain

1. The application of in situ hybridization histochemistry to the study of neuropeptide gene expression in human brain postmortem tissues is reviewed. We focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia. 32P-labeled oligonucleotides were used as hybridization probes. 2. Autoradiography combined with computerized image analysis was used to visualize and quantify the hybridization signal. 3. Several criteria were considered in order to ascertain the specificity of the signal, including Northern analysis, use of heterologous probes, competition assays, and thermal stability of the hybrids. 4. In control human striatum high levels of hybridization signal were observed for somatostatin, neuropeptide Y, and preproenkephalin A mRNAs. In contrast, no detectable signal was observed with the cholecystokinin, arginine-vasopressin, and oxytocin probes in this area. In the hypothalamus high levels of oxytocin and arginine-vasopressin mRNAs were visualized in several nuclei. Preproenkephalin A and somatostatin mRNAs were also observed in this region, while cholecystokinin mRNA was not detected. 5. No significant correlations were found between the density of the hybridization signal and parameters such as postmortem delay, age, and gender in the population studied. 6. Finally, alterations of mRNA levels for some of these peptides were found in Parkinson's disease and Huntington's chorea striatal tissues. 7. These results show that in situ hybridization histochemistry can be used to examine at the microscopic level neuropeptide gene expression in postmortem materials.     

Comparative analysis of estrogen receptor gene polymorphisms in apes

Polymorphic microsatellite repeats in the promoter region of estrogen receptor α gene (ESRα and the intron 6 region of estrogen receptor β gene (ESRβ) have been reported in human populations. To examine the evolutional state of both repeats, we surveyed the corresponding regions in DNA sequences from the following great apes and gibbons: 56 chimpanzees, 3 bonobos, 16 gorillas, 20 orangutans and 60 gibbons (four species: 17 of Hylobates agilis, 11 of H. lar, 15 of H. muelleri, and 17 of H. syndactylus). In the corresponding region of the TA repeat of human ESRα, chimpanzees and bonobos had two motifs in the repeat tract, (TA)_7–9 and (CA)_4–6. Gorillas had the (TA)_9–10 repeat tracts and orangutans had monomorphic (TA)₇ repeats. Although all great apes maintained the TA expansion, all gibbon sequences contained (TA)₂, implying that the CA dinucleotide expansion arose in the ancestor of chimpanzees and bonobos. The nucleotide sequences of ESRβ showed a very complex repeat pattern in apes. The human sequences had a non-variable preceding sequence at (CA) _n , (GA)₂(TA)₈(CA)₄(TA). In apes that region included {(TA) _n (CA) _n } _n . Gibbon sequences included (TATG) _n and (TATC) _n and no regular construction was observed. A deletion event in the reverse primer site seems to have occurred in the orangutan lineage. In addition, a great diversity of allele length was detected in each gibbon species.     

The effect of organic nitrogen and glucose on the production of recombinant human insulin-like growth factor in high cell densityEscherichia coli fermentations

Summary Two kinds of fed batch fermentation processes were compared at a 10-liter scale to examine their effect on recombinant human insulin-like growth factor (IGF-1) gene expression inEscherichia coli. The difference between the two processes was the feed medium composition and whether the process used a single or dual feed during the course of the fermentation. In the dual feed system, organic nitrogen was delivered at a higher rate (50 g/h) than in the single feed system (5 g/h). The dual feed process resulted in a significant increase in IGF-1 yield. 30 mg IGF-1/g dry cell weight was synthesized in the dual feed system compared to 3 mg IGF-1/g dry cell weight obtained in the single feed system. The presence of high levels of organic nitrogen during the induction period may enhance IGF-1 synthesis by protecting the IGF-1 from proteolytic degradation. The IGF-1 yield decreased to 17 mg/g dry cell weight when the glucose supply was decreased from 17 g/h to 8 g/h during the induction period; however, an increase in glucose supply from 17 g/h to 50 g/h during the induction period did not enhance the IGF-1 synthesis. Thus, the enhancement of IGF-1 gene expression in the dual feed process was mainly dependent on a high level of organic nitrogen and an appropriate level of glucose in the medium during the induction period.     

Identification of a novel c-DNA overexpressed in Fanconi's anemia fibroblasts partially homologous to a putative l-3-phosphoserine-phosphatase

We applied the cDNA differential display technique (DDT) in a DNA-repair deficient cell model to isolate genes involved in dysregulation of cell proliferation and development of cancer. The comparative analysis of mRNA expression patterns of human diploid fibroblasts from Fanconi's amemia (FA) and normal phenotype led to the identification of a novel cDNA CO9. Northern blot analysis reveals that CO9 is significantly upregulated in FA fibroblasts but downregulated or absent in fibroblasts from normal donors. CO9 was also highly expressed in FA B-cells of complementation group A and in Raji cells. However, CO9 is not expressed in FA complementation groups B, C, D and E. The full-length cDNA is 840 bp long and contains an open reading frame of 216 bp (72 amino acids), which encodes for a 7.6-kDa protein. The lengths of the 5′ and 3′ untranslated region are 165 and 459 bp, respectively. The N-terminal and C-terminal nucleotide sequence of CO9 shows homology to a putative human l-3-phosphoserine phosphatase identified recently (HSPSPASE, EMBL Accession No. Y10275) but lacks a 476-bp stretch in the open reading frame. The loss of nucleotides within the open reading frame introduces a new termination codon in the CO9 cDNA along with a novel COOH terminus resulting in a new protein product. Database chromosome mapping localized CO9 to chromosome 7q 11.2. We hypothesize that CO9 represents a novel protein being a partial homologue to the l-3-phosphoserine phosphatase but with a different regulatory cell function.