Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum

Abstract A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-α-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.     

Cloning and structure of the human interleukin 2 chromosomal gene.

Southern hybridization using 32P-labelled human interleukin 2 (IL2) cDNA probes revealed the existence of a single human IL2 gene. Five clones containing the human IL2 chromosomal gene were isolated from two different human DNA libraries cloned in either lambda Charon 4A or L47 phages. Analysis of the clones showed that they contained different, overlapping portions of human DNA which were derived from the same chromosomal segment. Restriction fragments which hybridized with labelled IL2 cDNA probes were subcloned into plasmid pUR250 and the sequence and organization of the IL2 gene was determined. It contains three introns, 90 bp, +/- 2400 bp and +/- 1900 bp in length, respectively. The organization of the genomic clone resembles that of another lymphokine, interferon-gamma, but no clear homology was found by comparing either the coding sequence or the 5'- and 3'-flanking sequences of the two genes.     

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.     

Isolation and characterization of serine protease gene(s) from Perkinsus marinus

This study reports the first serine protease gene(s) isolated from Perkinsus marinus. Using universal primers, a 518 bp subtilisin-like serine protease gene fragment was amplified from P. marinus genomic DNA and used as a probe to screen a lambda-phage P. marinus genomic library; 2 different lambda-phage clones hybridized to the digoxigenin(DIG)-labeled subtilisin-like gene fragment. Following subcloning and sequencing of the larger DNA fragment, a 1254 bp open reading frame was identified and later confirmed, by 5' and 3' random amplification of cDNA ends (RACE) and northern blot analysis, to contain the entire coding-region sequence. Sequence analysis of the 3' RACE results from 2 isolate cultures, VA-2 (P-1) and LA 10-1, revealed multiple polymorphic sites within and among isolates. We identified 2 different types of cDNA clones with 95.53% nucleotide sequence similarity, suggesting the possibility of 2 closely related genes within the P. marinus genome. Southern blot analysis of genomic DNA from 12 genetically distinct P. marinus isolate cultures revealed 2 different banding patterns among isolates.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

The rat interleukin-5 gene: characterization and expression by retroviral gene transfer and polymerase chain reaction.

The rat interleukin-5 (IL-5) gene was isolated from a genomic lambda phage library and a fragment containing all four exons was inserted into the retroviral vector pXT1, resulting in pXTRIL5. Upon retroviral gene transfer into two IL-5-dependent mouse cell lines, B13 and T88M, autonomously growing cells were established and B-cell growth factor activity was detected in the supernatants of the infected cells. "cDNA" versions of the rat IL-5 gene were rescued by the polymerase chain reaction (PCR) with primers specific for the flanking regions of the cloning site in pXT1. Restriction or DNA sequence analysis of five different clones revealed precise splicing in two cases, while three of the clones had retained the first intron. In addition, in two of these about 400 bp of rat IL-5 5' flanking regions were deleted. The sequence comparison of rat, mouse, and human IL-5 genes revealed a high degree of conservation (e.g., mouse and rat were 92% homologous at the amino acid level). The combination of retroviral gene transfer and PCR may offer an alternative, efficient method for the cloning of cDNAs.     

Cloning and nucleotide sequence of the murine interleukin-3 gene

Southern hybridization analysis using a probe derived from a murine interleukin-3 (IL-3) cDNA clone revealed the presence of a single IL-3 gene in the haploid murine genome. An 8600-base-pair (8.6-kb) murine genomic EcoRI fragment containing the IL-3 gene was isolated by screening a library of size-fractionated genomic EcoRI fragments cloned in lambda gtWES X lambda B. The nucleotide sequence of a 3.5-kb region of the cloned DNA encompassing the IL-3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14-15-bp tandem repeating sequence which resembles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL-3 cDNA cloned from WEHI-3, a tumorigenic cell line which over-produces IL-3, establishing that the unprocessed primary structure of IL-3 is identical in WEHI-3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL-3 gene in WEHI-3 cells.     

Cloning of interleukin 2 mRNAs from human tonsils

Human interleukin 2 (IL-2) mRNAs were cloned from a cDNA library prepared from mitogen-stimulated tonsillar mononuclear cells. One of these clones was sequenced and, using this cDNA as a hybridization probe, Southern blot analysis of the human placental DNA was performed. Our results indicate that there is only one IL-2 gene in the human genome.     

Molecular biology of the alpha-L-fucosidase gene and fucosidosis

Human alpha-L-fucosidase, a lysosomal enzyme, hydrolyzes alpha-L-fucose from glycolipids and glycoproteins. Its activity is deficient in human fucosidosis an autosomal recessive disease. In order to understand the molecular basis of this lysosomal storage disorder we have cloned several cDNAs coding for human alpha-L-fucosidase from a human hepatoma and a human liver cDNA library constructed in lambda gt11. Compiling the cDNA sequences of these clones we have identified 1,829 base pairs (bp) encoding human alpha-L-fucosidase. This includes an open reading frame of 1,172 bp, a consensus polyadenylation signal AAT AAA and a poly(A)+ tail. The sequence is incomplete at the 5'-end, and clones encoding the amino terminus of the native protein, the propeptide and leader signal have not yet been isolated. The open reading frame encodes for 390 amino acids with a calculated Mr of 45,557. This represents 78-95% of the mature processed alpha-L-fucosidase. The availability of these cDNA clones has enabled us to map the structural gene for alpha-L-fucosidase to chromosome 1p34.1-1p36.1 by Southern blot analysis of DNA from human-rodent somatic cell hybrids and by in situ hybridization. Furthermore, a Pvu II restriction fragment length polymorphism (RFLP) has been identified at the human alpha-L-fucosidase gene locus. Analysis of mRNA by Northern blotting gives a major species of 2.25 kb. In 4 patients with fucosidosis no mRNA signal was detected and Western blots gave no immunoreactive enzyme. Southern blotting after Eco RI digestion in two fucosidosis families revealed a banding abnormality (extra 6-kb band).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.     

Comparative analysis of Xenopus tropicalis and Xenopus laevis vitellogenin gene sequences

Analysis of cDNA clones synthesized from vitellogenin mRNA of X. tropicalis revealed three different types of cDNA clones, i.e. A, A* and B. A and A* clones have a sequence divergence of about 6% and are both related to X. laevis vitellogenin cDNAs of subgroup A1 as well as A2 with a sequence divergence of 6-9%. B clones however, are related to X. laevis cDNA clones of subgroup B1 and B2 with a sequence divergence of about 7%. While the A and B clones correspond to vitellogenin mRNAs of similar abundance, A* clone is complementary to a vitellogenin mRNA about 100 fold less abundant than A and B mRNAs although all three vitellogenin mRNAs are encoded by single copy genes. Furthermore, two forms of A* mRNA were found. One of the two is lacking an internal fragment of about 900 bp. Since this DNA fragment is highly repeated in the genome, we suggest that this A* clone was synthesized from a processing intermediate of the A* precursor vitellogenin mRNA.     

Molecular biology of Diazepam Binding Inhibitor peptide

Complementary DNA (cDNA) clones containing the entire coding sequence for Diazepam Binding Inhibitor (DBI) peptide, a 10-kDa precursor of putative natural ligands of benzodiazepine recognition sites, were isolated from rat, human and cow libraries. The sequence of all these clones is highly conserved; however, the N-terminal sequence predicted by the human DBI clone differed from that of the other two clones. DBI cDNA, utilized as hybridization probe in Southern blot analysis, revealed that DBI of both human and rat might be encoded by a multiple family of 4–6 genes. Furthermore, we have used in situ chromosomes hybridization to map human DBI genes. The results indicate that a human DBI gene is localized on chromosome 2 and that three of the four hybridization signals detected by the human DBI probe are located on three other chromosomes. These findings raise a question as whether multiple DBI genes encode for different molecular forms of DBI. In the attempt to test this hypothesis, cow cDNA and human genomic libraries were screened with DBI cDNA. In this paper I report the isolation of clones from these libraries which, although hybridizing well to DBI cDNA, possess a low percentage of homology (46.7%), randomly distributed within the coding region of DBI cDNA. Whether or not these clones encode for peptides sharing the same physiological role as DBI is under investigation.Special issue dedicated to Dr. Erminio Costa     

人白细胞介素6cDNA克隆的分离与鉴定

我们从重组的人α干扰素处理的单层HeLa细胞常规提取Poly(A)~+RNA作为逆转录合成cDNA第一链之模板,用引物-适配接头法在噬菌质粒pTz19R中构建cDNA文库。以~(32)p-标记的480bpIL-6cDNA片段作探针进行菌落原位及狭缝印迹杂交,筛选出6个阳性克隆。其中两个克隆并用限制酶切分析及DNA序列测定做进一步鉴定。结果证明,一个克隆的cDNA片段长1.3kb,含有人白介素6全长编码区;另一个的cDNA插入片段为0.9kb,缺乏信号肽及成熟IL-6N端30个残基的编码序列。     

Identification of two processed pseudogenes of the human Tom20 gene

The open reading frame (ORF) of the human Tom20 gene (hTom20) was amplified by PCR from a HeLa cDNA library using primers based on the sequence of HUMRSC145 and cloned into a pET15b vector. Amplification of human genomic DNA using these primers yielded a DNA fragment of the same size as that of the ORF of hTom20 cDNA. Sequencing of this fragment revealed that: (1) it has the same number of base pairs as the ORF of hTom20 cDNA (438 bp); and (2) the two sequences differ by 14 single base pair substitutions (97% similarity) causing eight changes in the amino acid sequence and two premature stop codons. Further amplification of human genomic DNA adaptor-ligated libraries using primers based on HUMRSC145 revealed three different sequence-related genomic regions; one corresponding to the fragment referred above, another corresponding to the hTom20 gene, and a third fragment of which the sequence differs from the ORF of hTom20 cDNA by only 22 base pair substitutions and a deletion of 4 bp. We conclude that, in addition to the hTom20 gene, there are two genomic DNA sequences (Ψ1Tom20 and Ψ2Tom20) that are processed pseudogenes of hTom20. Aspects concerning their evolutionary origin are discussed. Received: 12 September 1997 / Accepted: 29 November 1997     

cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase

A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes. This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method. Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively. The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes. ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes. The probe also detected a sequence on chromosome 22. Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.     

Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication.

The temperature-sensitive ts A1S9 mutation of mouse L cells was previously shown to affect nuclear DNA replication and to be complemented by active and inactive human X chromosomes in human-ts A1S9 somatic cell hybrids. We report the isolation of cDNA clones which correct the ts A1S9 lesion, using as a probe a genomic fragment derived from the human A1S9 locus. The nucleotide sequence of the A1S9 cDNA encompasses a single open reading frame of 2409 bp which could encode a heretofore unreported protein of 90 393 daltons. Southern blot hybridization of the A1S9 cDNA probe with DNA from various species revealed homologous sequences in vertebrates but not in yeast. Northern blot analysis of serum-starved, synchronized cells demonstrated that the A1S9 gene was expressed at a relatively low level in quiescent cells and at a higher and constant level throughout the cell cycle. Human cell lines harbouring increasing numbers of inactive X chromosomes (47, XXX, 49, XXXXX) were found to express the A1S9 gene at the same level as control cells (45, X), suggesting that the gene does not escape X chromosome inactivation.