Jerry L. Slightom

The structure and evolution of the human β-globin gene family

Argiris Efstratiadis Department of Biological Chemistry Harvard Medical School Boston, Massachusetts 02115 James W. Posakony, Tom Maniatis, Richard M. Lawn* and Catherine O’Connell+ Division of Biology California Institute of Technology Pasadena, California 91125 Richard A. Spritz, Jon K. DeRiel,# Bernard G. Forget and Sherman M. Weissman Departments of Genetics and Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510 Jerry L. Slightom, Ann E. Blechl and Oliver Smithies Laboratory of Genetics University of Wisconsin Madison, Wisconsin 53706 Francisco E. Baralle, Carol C. Shoulders and Nicholas J. ProudfootQ MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England Summary We present the results of a detailed comparison of the primary structure of human p-like globin genes and their flanking sequences. Among the se- quences located 5’ to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 2 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. Calcula- tion of the divergence times of individual @like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence com- parisons. In addition, the evolutionary relationship of the embryonic e-globin gene to the other human P-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 B-like globin genes during evolution.

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.

Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors.

Sequences coding for the bean seed protein phaseolin were inserted into transferred DNA regions of tumor-inducing plasmids. Constructions were devised in which the coding region of phaseolin was fused in the correct reading frame with the coding region of octopine synthase and placed under the transcriptional control of the octopine synthase promoter. Other plasmids were prepared to permit expression of the phaseolin-encoding sequences from the flanking phaseolin promoter region. The RNA transcribed in sunflower cells transformed with these constructions was characterized by hybridization procedures, SI nuclease mapping, and by translation in vitro of extracted RNA. These tests showed that the genomic intervening sequences were correctly excised. Immunoreactive phaseolin polypeptides were detected by enzyme-linked immunosorbent assay and by antibody hybridization to electrophoretically separated protein extracts of sunflower tissues isolated from crown gall tumors and of transformed sunflower cells grown in tissue culture. These results demonstrate the expression of a plant gene after transfer to a taxonomically distinct botanical family.

A human homolog of the yeast CDC7 gene is overexpressed in some tumors and transformed cell lines

The Cdc7 protein kinase of Saccharomyces cerevisiae is a critical regulator of several aspects of DNA metabolism and cell cycle progression. We describe the isolation of a human gene encoding a Cdc7 homolog. The Cdc7Hs protein sequence is 27% identical to that of the yeast protein, includes features unique to yeast Cdc7, and contains all conserved catalytic residues of protein kinases. The human sequence also shows significant similarity to the cyclin-dependent kinases, in accordance with evidence that yeast Cdc7 is related to the cdks. CDC7Hs is expressed in many normal tissues, but overexpressed in certain tumor types and all transformed cell lines examined. In some of the tumors tested, CDC7Hs expression correlates with expression of a proliferation marker, the histone H3 gene. In other cases, no such correlation was observed. This suggests that CDC7Hs expression may be associated hyperproliferation in some tumors and neoplastic transformation in others.

Size and organization of a multigene family encoding phaseolin, the major seed storage protein of Phaseolus vulgaris L.

SummarySolution hybridization kinetics and genomic nitrocellulose blot hybridization analyses show that the Phaseolus vulgaris L. (French bean) storage proteins (phaseolins) are encoded as a small, homologous, multigene family consisting of approximately seven members. Restriction endonuclease site mapping (EcoRI, BamHI, and BglII) of DNA regions flanking the phaseolin genes has shown that the gene family can be divided into at least three characteristic fragment size classes. Clones representative of two of these phaseolin gene classes have been isolated from a λ1059 phage library.

Base substitutions, length differences and DNA strand asymmetries in the human Gγ and Aγ fetal globin gene region

Abstract We have studied differences arising subsequent to the 5 kilobase pair (kb) duplication that led to the human G γ and A γ 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 β-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 ≥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.

Efficient functional coupling of the human D3 dopamine receptor to Go subtype of G proteins in SH-SY5Y cells

The D3 dopamine receptor presumably activates Gi/Go subtypes of G‐proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non‐neuronal cell lines. In this study, recombinant human D3 receptors expressed in a human neuroblastoma cell line, SH‐SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin‐stimulated cyclic AMP production and Ca2+‐channel (N‐type) currents in SH‐SY5Y cells, and enhanced GTPγ35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. GTPγ35S‐bound Gα subunits from quinpirole‐activated and solubilized membranes were monitored upon immobilization with various Gα‐specific antibodies. Gαo subunits (not Gαi) were highly labelled with GTPγ35S in SH‐SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription‐polymerase chain reaction and Western blots. N‐type Ca2+ channels and adenylyl cyclase V (D3‐specific effector), on the other hand, exist only in SH‐SY5Y cells. More efficient coupling of the D3 receptor to Go subtypes in SH‐SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH‐SY5Y cells (N‐type Ca2+‐channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor.

Isolation and identification of TL-DNA/plant junctions in Convolvulus arvensis transformed by Agrobacterium rhizogenes strain A4.

We have constructed a Charon 4A phage library containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant (clone 7) regenerated from a root organ culture incited by Agrobacterium rhizogenes, strain A4. Using a subcloned region of the Ri plasmid as 32P‐labeled probe, two lambda clones containing most of the ‘left’ T‐DNA (TL) region were isolated. One of these lambda clones contains the left TL‐DNA/plant junction, which was located by comparing nucleotide sequences from the appropriate regions of the Ri plasmid and this lambda clone. A 25‐bp sequence found near this left TL‐DNA/plant junction matches the 25‐bp terminal sequence found at or near T‐DNA/plant junctions of both nopaline‐ and octopine‐type A. tumefaciens Ti plasmids. A possible location for the right Ri TL‐DNA/plant junction in C. arvensis clone 7 was found by obtaining the nucleotide sequence surrounding its mapped location. Hybridization of plant DNA found adjacent to the left TL‐DNA/plant junction against total C. arvensis DNA shows that this T‐DNA integration occurred in a plant DNA region that does not contain highly repetitive DNA sequences. Nucleotide sequence analysis of 1004 bp of this plant DNA revealed no complete or partial open reading frames, but this plant DNA does have the potential to form various secondary structures which might play a role in the T‐DNA integration event.

Differential pharmacology between the guinea-pig and the gorilla 5-HT1D receptor as probed with isochromans (5-HT1D-selective ligands).

Both the 5‐HT1D and 5‐HT1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5‐HT1D receptors with much higher affinity than 5‐HT1B receptors. In the guinea‐pig, a primary animal model for anti‐migraine drug testing, however, isochromans bound the 5‐HT1D receptor with lower affinity than the gorilla receptor. This species‐specific pharmacology was investigated, using site‐directed mutagenesis on cloned guinea‐pig receptors heterologously expressed in human embryonic kidney 293 cells. Mutations of threonine 100 and arginine 102 at the extracellular side of transmembrane II of the guinea‐pig 5‐HT1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its affinity for isochromans to that of the gorilla receptor, with little effects on its affinities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. For G protein‐coupling, serotonin marginally enhanced GTPγ35S binding in membranes expressing the guinea‐pig 5‐HT1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPγ35S binding in the latter nearly as much as serotonin, and several isochromans by 30–60% of serotonin. We discovered key differences in the function and binding properties of guinea‐pig and gorilla 5‐HT1D receptors, and identified contributions of I100 and H102 of primate 5‐HT1D receptors to isochroman binding. Among common experimental animals, only the rabbit shares I100 and H102 with primates, and could be useful for studying isochroman actions in vivo.

Agrobacterium tumefaciens T-DNA integrates into multiple sites of the sunflower crown gall genome

SummaryWe analyzed the integration of a tumor inducing (Ti) plasmid into an octopine producing crown gall tumor of sunflower, line PSCG 15955. A continuous Ti plasmid segment (T-DNA) of about 19.5 kilo base pairs (kbp) is transferred and integrated into a small number of sites of the plant DNA.The number of T-DNA integration sites in our tumor line was estimated by two different methods. First, cloned fragments of the T-DNA were hybridized to tumor DNA and the hybridization patterns were observed. The number of T-DNA integration sites in line PSCG 15955 was found to be approximately eight.Second, a library was constructed from total DNA of tumor line PSCG 15955 by molecular cloning using the bacteriophage lambda vector Charon 4A. Recombinant phages having sequence homologies to the Ti plasmid were selected. The lower limit on the number of integration sites was three because we obtained three different right hand side genomic clones of plant/T-DNA hybrids. The initial screening of the library also revealed two left hand border clones. Hybridization of these five distinct recombinant clones to uninfected sunflower DNA shows that the cloned T-DNA segments are covalently bonded to plant DNA. The left and right hand plant boundary sequences are homologous to either unique and or repeated plant DNA segments.