Biomedical subjects

J L Slightom

Publications and source records attributed to J L Slightom.

106 records · Page 6Linked to original sources

PubMed1985

Expression of phaseolin cDNA genes in yeast under control of natural plant DNA sequences.

We have constructed a strain of Saccharomyces cerevisiae that expresses two different members of the multigene family encoding phaseolin, the major seed storage glycoprotein from the French bean, Phaseolus vulgaris. Yeast vector plasmids have been engineered to include a Phaseolus DNA segment that contains the natural 5' and 3' plant genomic regulatory sequences flanking a cDNA copy of the protein-encoding region. Characterization of phaseolin transcripts isolated from transformed yeast cells revealed the presence of two classes of polyadenylylated RNA, approximately 1400 and 1800 bases, which initiate and terminate in plant DNA sequences. Protein extracts from transformants contain phaseolin-immunoreactive proteins similar in size to those isolated from plant tissue. These polypeptides are glycosylated in yeast and their molecular weights are consistent with the possibility that the phaseolin signal peptide has been cleaved to form the mature protein.

Journal Article↗

PubMed1985

Nucleotide sequence, evolution, and expression of the fetal globin gene of the spider monkey Ateles geoffroyi.

The single gamma-globin gene of the New World spider monkey Ateles geoffroyi is similar to other primate genes of the beta-globin gene cluster ("beta-like" globin genes). The number of nonsynonymous nucleotide substitutions between the coding regions of Ateles and other primate gamma-globin genes suggests that the Platyrrhine and Catarrhine evolutionary lines diverged approximately equal to 40 million years ago, an estimate reasonably consistent with the fossil record. However, the number of synonymous coding region and noncoding base differences is much smaller than predicted by various molecular "clocks." This suggests that the rate of synonymous coding and noncoding base substitution has not been constant per absolute time in primate lineages. Expression of the cloned Ateles gamma-globin gene in cultured monkey cells showed that the sequence AAUAAA near the mRNA 3' terminus is not sufficient to define the site of transcript polyadenylylation.

Amino Acid Sequence↗

PubMed1985

Chimpanzee fetal G gamma and A gamma globin gene nucleotide sequences provide further evidence of gene conversions in hominine evolution.

The fetal globin genes G gamma and A gamma from one chromosome of a chimpanzee (Pan troglodytes) were sequenced and found to be closely similar to the corresponding genes of man and the gorilla. These genes contain identical promoter and termination signals and have exons 1 and 2 separated by the conserved short intron 1 (122 bp) and exons 2 and 3 separated by the more rapidly evolving, larger intron 2 (893 bp and 887 bp in chimpanzee G gamma and A gamma, respectively). Each intron 2 has a stretch of simple sequence DNA (TG)n serving possibly as a "hot spot" for recombination. The two chimpanzee genes encode polypeptide chains that differ only at position 136 (glycine in G gamma and alanine in A gamma) and that are identical to the corresponding human chains, which have aspartic acid at position 73 and lysine at 104 in contrast to glycine and arginine at these respective positions of the gorilla A gamma chain. Phylogenetic analysis by the parsimony method revealed four silent (synonymous) base substitutions in evolutionary descent of the chimpanzee G gamma and A gamma codons and none in the human and gorilla codons. These Homininae (Pan, Homo, Gorilla) coding sequences evolved at one-tenth the average mammalian rate for nonsynonymous and one-fourth that for synonymous substitutions. Three sequence regions that were affected by gene conversions between chimpanzee G gamma and A gamma loci were identified: one extended 3' of the hot spot with G gamma replaced by the A gamma sequence, another extended 5' of the hot spot with A gamma replaced by G gamma, and the third conversion extended from the 5' flanking to the 5' end of intron 2, with G gamma replaced here by the A gamma sequence. A conversion similar to this third one has occurred independently in the descent of the gorilla genes. The four previously identified conversions, labeled C1-C4 (Scott et al. 1984), were substantiated with the addition of the chimpanzee genes to our analysis (C1 being shared by all three hominines and C2, C3, and C4 being found only in humans). Thus, the fetal genes from all three of these hominine species have been active in gene conversions during the descent of each species.

Animals↗

PubMed1984

Isolation and nucleotide sequence analysis of the beta-type globin pseudogene from human, gorilla and chimpanzee.

The beta-globin gene cluster of human, gorilla and chimpanzee contain the same number and organization of beta-type globin genes: 5'-epsilon (embryonic)-G gamma and A gamma (fetal)-psi beta (inactive)-delta and beta (adult)-3'. We have isolated the psi beta-globin gene regions from the three species and determined their nucleotide sequences. These three pseudogenes each share the same substitutions in the initiator codon (ATG----GTA), a substitution in codon 15 which generates a termination signal TGG----TGA, nucleotide deletion in codon 20 and the resulting frame shift which yields many termination signals in exons 2 and 3. The basic structure of these psi beta-globin genes, however, remains consistent with that found for functional beta-globin genes: their coding regions are split by two introns, IVS 1 (which splits codon 30, 121 base-pairs in length) and IVS 2 (which splits codon 104, 840 to 844 base-pairs in length). These introns retain the normal splice junctions found in other eukaryotic split genes. The three hominoid psi beta-globin genes show a high degree of sequence correspondence, with the number of differences found among them being only about one-third of that predicted for DNA sites evolving at the neutral rate (i.e. for sites evolving in the absence of purifying selection). Thus, there appears to be a deceleration in the rate of evolution of the psi beta-globin locus in higher primates.

Animals↗

PubMed1984

Use of a recombination-deficient phage lambda system to construct wheat genomic libraries.

The poor cloning efficiency of wheat (Triticum aestivum cv. Yamhill) DNA in conventional cloning vectors has previously prevented the preparation of complete genomic libraries. We show here that while wheat DNA does not clone efficiently using the vector Ch4A, it can be cloned efficiently using Ch32. Ch32 clones are red- gam+ and therefore can be propagated on recombination-deficient hosts. These results suggest that instability of wheat sequences in conventional lambda vector systems has frustrated previous attempts to prepare libraries.

Bacteriophage lambda↗

PubMed1984

The sequence of the gorilla fetal globin genes: evidence for multiple gene conversions in human evolution.

Two fetal globin genes (G gamma and A gamma) from one chromosome of a lowland gorilla (Gorilla gorilla gorilla) have been sequenced and compared to three human loci (a G gamma-gene and two A gamma-alleles). A comparison of regions of local homology among these five sequences indicates that long after the duplication that produced the two nonallelic gamma-globin loci of catarrhine primates, about 35 million years (Myr) ago, at least one gene conversion event occurred between these loci. This conversion occurred not long before the ancestral divergence (about 6 Myr ago) of Homo and Gorilla. After this ancestral divergence, a minimum of three more gene conversion events occurred in the human lineage. Each human A gamma-allele shares specific sequence features with the gorilla A gamma-gene; one such distinctive allelic feature involves the simple repeated sequence in IVS 2. This suggests that early in the human lineage the A gamma-genes may have undergone a crossing-over event mediated by this simple repeated sequence. The DNA sequences from coding regions of both G gamma- and A gamma-loci, a comparison of 292 codons in the corresponding gorilla and human genes, show an unusually low evolutionary rate, with only two nonsilent differences and, surprisingly, not even one silent substitution. The two nonsynonymous substitutions observed predict a glycine at codon 73 and an arginine at codon 104 in the gorilla A gamma-sequence rather than aspartic acid and lysine, respectively, in human A gamma. Because only arginine has been found at position 104 in gamma-chains of Old World monkeys, it may represent the ancestral residue lost in gorilla and human G gamma-chains and in the human A gamma-chain. Possibly the arginine codon (AGG) was replaced by the lysine codon (AAG) in the G gamma-gene of a common ancestor of Homo and Gorilla and then was transferred to the A gamma-gene by subsequent conversions in the human lineage. DNA sequence conversions, similar to that attributed to the fetal gamma-globin genes, appear to be relatively frequent phenomena and, if widespread throughout the genome, may have profound evolutionary consequences.

Alleles↗

PubMed1983

Complete nucleotide sequence of a French bean storage protein gene: Phaseolin.

The complete nucleotide sequences of the gene and the mRNA coding for a specific phaseolin type French bean major storage protein have been determined. Comparison of these sequences reveals a phaseolin gene structure consisting of 80 base pairs (bp) of 5' untranslated DNA, 1,263 bp of protein-encoding DNA which is interrupted by five intervening sequences (IVS1, 72 bp; IVS2, 88 bp; IVS3, 124 bp; IVS4, 128 bp; and IVS5, 103 bp), and 135 bp of 3' untranslated DNA. Sequences characteristic of eukaryotic promoters "CCAAT" and "TATA" are present in the 5' flanking DNA, and the eukaryotic poly(A) addition signal A-A-T-A-A-A occurs 16 bp before the first nucleotide of poly(A). The derived amino acid sequence yields an amino acid composition and a molecular weight compatible with those found for the beta-type phaseolin protein. Two regions that probably serve as carbohydrate-peptide linkage recognition sites have been identified. A region of highly hydrophobic amino acids at the NH(2) terminus of the protein suggests the presence of a signal peptide in the newly synthesized phaseolin protein.

Journal Article↗

PubMed1981

Base substitutions, length differences and DNA strand asymmetries in the human G gamma and A gamma fetal globin gene region.

We have studied differences arising subsequent to the 5 kilobase pair (kb) duplication that led to the human G gamma and A gamma fetal globin genes. The local occurrence of base substitutions in the duplicated 5 kb region correlates positively with the local AT base pair content. This correlation also occurs in two mouse beta-globin genes and in two mouse immunoglobulin genes. The relationship is valid for transcribed or nontranscribed DNA and for DNA that contains only coding sequences. Length differences in the fetal globin duplicated regions correlate positively with the occurrence of short direct repeats of greater than or equal 5 base pairs. Path analysis of the interrelationships of base composition, base substitutions, repeats and length differences provides an integrated view of the relative effects on chromosomal changes of these variables and of selection. The distributions along the chromosome of simple sequences and of base compositions show highly significant local asymmetries between the transcribed and nontranscribed strands of the DNA, which permit us to divide the fetal globin gene region into chromosomal domains. Comparable domains are present in DNA from other sources, including the mammalian viruses SV40 and polyoma virus strain A-2 in which some of the domains appear related to discrete functions.

Animals↗

PubMed1981

A history of the human fetal globin gene duplication.

The nucleotide sequence of 11.4 kilobase pairs (kb) of human DNA that includes the two fetal globin genes, G gamma and A gamma, shows that they are part of a 5 kb tandem duplication. A small segment of DNA occurs three times, on either side of and between the two duplicates. We present two models that account for these observations. One model is simple but requires the assumption of a preexisting repetitive element; the other is more complex but does not require the assumption of preexisting repeats. Over much of the 5 kb duplicated region, the present duplicate copies differ by an average of 14% of their bases, from which we calculate that the duplication was first formed about 34 million years ago. However, 1.5 kb of DNA are vitually identical in the two genes analyzed here, probably as a consequence of an intergenic exchange (gene conversion) that replaced part of the diverging A gamma gene with the corresponding part of the G gamma gene. This conversion took place around 1 million years ago. A sequence of repeated dinucleotides may be one signal involved in exchanges leading to this and similar conversions. Cycles of duplication, triplication, deletion and gene conversion on probably common in many multigene families.

Base Sequence↗

PubMed1981

Mouse IgA heavy chain gene sequence: implications for evolution of immunoglobulin hinge axons.

The complete nucleotide sequence of the gene and mRNA coding for the constant (C) region of the secreted form of the BALB/c mouse IgA immunoglobulin alpha heavy (H) chain has been determined. As in other immunoglobulins, the three C region domains of the alpha protein, C alpha 1, C alpha 2, and C alpha 3 are coded in separate exons. However, the hinge region of C alpha is not coded on a separate exon as it is in other hinge-containing immunoglobulins. Instead, the alpha hinge is coded as a 5' extension of the C alpha 2 exon, and we suggest that it may have evolved by duplication leading to incorporation of an acceptor RNA splice site into the coding portion of the C alpha 2 exon. Extensions of this concept could provide an explanation for duplications in the human alpha 1 chain.

Amino Acid Sequence↗

PubMed1980

Human fetal G gamma- and A gamma-globin genes: complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes.

We present the nucleotide sequences of the G gamma-and A gamma-globin genes from one chromosome (A) and of most of the A gamma gene from the other chromosome (B) of the same individual. All three genes have a small, highly conserved intervening sequence (IVS1) of 122 bp located between codons 30 and 31 and a large intervening sequence (IVS2) of variable length (866-904 bp) between codons 104 and 105. A stretch of simple sequence DNA occurs in IVS2 which appears to be a hot spot for recombination. On the 5' side of this simple sequence, the allelic A gamma genes differ considerably in IVS2 whereas the nonallelic G gamma and A gamma genes from chromosome A differ only slightly. Yet on the 3' side of the simple sequence, the allelic genes differ only slightly whereas the nonallelic genes differ considerably. We hypothesize that the 5' two thirds of the A gamma gene on chromosome A has been "converted" by an intergenic exchange to become more like the G gamma gene on its own chromosome A than it is like the allelic A gamma gene on the other chromosome B. Our sequence data suggest that intergenic conversions occur in the germ line. The DNA sequence differences between two chromosomes from a single individual strongly suggest that DNA sequence polymorphisms for localized deletions, additions and base substitutions are very common in human populations.

Alleles↗

PubMed1980

The structure and evolution of the human beta-globin gene family.

We present the results of a detailed comparison of the primary structure of human beta-like globin genes and their flanking sequences. Among the sequences located 5' to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 +/- 1 and 77 +/- 10 bp, respectively, 5' to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calculation of the divergence times of individual beta-like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence comparisons. In addition, the evolutionary relationship of the embryonic epsilon-globin gene to the other human beta-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of beta-like globin genes during evolution.

Animals↗

PubMed1979

Structure of the constant and 3' untranslated regions of the murine gamma 2b heavy chain messenger RNA.

The complete coding sequence for the constant region of the mouse gamma 2b immunoglobulin heavy chain and the 3' untranslated region has been determined. The coding portion of the sequence is 1008 nucleotides long (amino acid residues 114 to 449), and the 3' noncoding region contains 102 nucleotides preceeding the polyadenylate. An extra carboxyl-terminal lysine residue which had not been observed in the gamma 2b or other gamma subclass protein sequences occurs in the nucleotide sequence and is probably processed posttranslationally. A 17-nucleotide sequence occurs with slight variation twice in CH1 and once in CH2 domains in the same relative location but with different translational phase. This sequence may be the site of crossover in a gamma 2b . gamma 2a heavy chain variant, an indication of possible recombinational activity of some kind.

Animals↗

PubMed1978

Cloning human fetal gamma globin and mouse alpha-type globin DNA: preparation and screening of shotgun collections.

Shotgun collections of Charon 3A bacteriophages containing Eco RI fragments of human and mouse DNA were constructed with the use of in vitro packaging. Plaques were screened by hybridization, and globin-specific clones were isolated from both human (Charon 3AHs51.1) and mouse (Charon 3AMm30.5). The fragments cloned were detected in unfractionated genomic DNA by the Southern method of hybridization.

Animals↗

PubMed1978

Cloning human fetal gamma globin and mouse alpha-type globin DNA: characterization and partial sequencing.

Two globin-related clones isolated from collections of bacteriophages containing unfractionated Eco RI fragments of human and mouse DNA were characterized. Charon3AHs51.1Hbgamma includes 2.7 kilobase pairs of human DNA containing a large part of a fetal gamma globin chain structural gene; Charon 3AMm30.5 includes 4.7 kilobase pairs of mouse DNA related to alpha globin. The human fetal gamma globin gene has within its coding region two intervening sequences of noncoding DNA, IVS 1 and IVS 2, of approximately 1-0 and 900 base pairs. Sequence IVS 1 is located at the position of one of the two intervening sequences occurring in adult globin genes; IVS 2 is located at the position of the other.

Amino Acid Sequence↗

PubMedDate not supplied

Primate eta-globin DNA sequences and man's place among the great apes.

Molecular studies indicate that chimpanzee and gorilla are the closest relatives of man (refs 1-7 and refs therein). The small molecular distances found point to late ancestral separations, with the most recent being between chimpanzee and man, as judged by DNA hybridization. Kluge and Schwartz contest these conclusions: morphological characters group a chimpanzee-gorilla clade with the Asian ape orang-utan in Kluge's cladistic study and with an orang-utan-human clade in Schwartz's study. Clearly, extensive sequencing of nuclear DNA is needed to resolve by cladistic analysis the branching order within Hominoidea. Towards this goal, we are sequencing orthologues of the primate psi eta-globin locus. Here, we compare the newly completed sequences of orang-utan and rhesus monkey with human, chimpanzee, gorilla, owl monkey, lemur and goat orthologues. Our findings substantially increase the evidence indicative of a human-chimpanzee-gorilla clade with ancestral separations around 8 to 6 Myr ago. We also verify that neutral hominoid DNA evolved at markedly retarded rates.

Animals↗